Novel peptidic linkers and cryptophycin conjugates, their preparation and their therapeutic use

ABSTRACT

wherein RCG1 represents a reactive chemical group being reactive towards a chemical group present on a polypeptide such as an antibody; P represents H, OH or an activated O; and L represents a specific linker. The disclosure also relates to cryptophycin payloads, as well as to cryptophycin conjugates, to compositions containing them and to their therapeutic use, especially as anticancer agents. The disclosure also relates to the process for preparing these conjugates.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/975,423, filed May 9, 2018, which claims priority to European PatentApplication No. 17305531.0, filed May 10, 2017, the entirety of which ishereby incorporated herein by reference.

The present disclosure relates to new peptidic linkers, to newcryptophycin payloads, to new cryptophycin conjugates, to compositionscontaining them and to their therapeutic use, such as for use asanticancer agents. The present disclosure also relates to the processfor preparing these conjugates.

These linkers are enzymatically cleaved in the lysosome of cells, byenzymes such as Cathepsin B for example. Cathepsin B is a cysteineproteinase belonging to the papain family and one of the main lysosomalproteinases among mammals. It is involved in protein turnover and in themaintenance of cellular metabolism as well as in several otherphysiological or pathological processes, like for example tumoralprogression. Its over-expression, both at the genetic and proteiclevels, has been demonstrated in tumors, the increase of protein leadingalso to an increase of enzymatic activity. This was the basis of severalprodrug approaches of chemotherapeutic agents (Int J Oncology 2013, 42,373-383) but also of the conception of cleavable linkers forantibody-drug conjugates (ADC) (Bioconj Chem 2002, 13, 855-869). ForADC, one commonly used peptidic linker consists of the sequenceValine-Citrulline (ValCit) joined to the p-aminobenzylic alcoholself-immolative moeity (J Org Chem 2002, 67, 1866-1872), as exemplifiedin WO2011/001052 with example 23. Associated to a cytotoxic drug, thesubsequent construct is quite hydrophobic which can be challenging forachieving certain drug-to-antibody ratio (DAR), monomeric purity and ADCstability. Actually, despite extensive conjugation optimization, example23 of WO2011/00152 couldn't be successfully conjugated to provide an ADCwith satisfactory DAR and monomeric purity, namely DAR >2 and >95% ofmonomers.

Since drugs used for ADC—like tubulin or DNA binders—are essentiallyhydrophobic, there was a need for more hydrophilic peptidic linkers toimprove the solubility of the payload—a payload is a compound comprisingthe cytotoxic drug conjugated to a linker—and thus potentially itsreactivity towards antibody conjugation and the stability of thesubsequent ADC. Increasing payload solubility should allow to increasethe DAR, the monomeric purity and ADC stability, especially in terms ofaggregation propensity.

SUMMARY OF THE DISCLOSURE

The disclosure relates to new peptidic compounds, chosen from compoundsof formula (I):

RCG1-L-P  (I)

wherein

-   -   RCG1 represents a reactive chemical group that is reactive        towards a chemical group present on a polypeptide such as an        antibody;    -   P represents a hydrogen atom, —OH or an activated O wherein an        activated O is defined below.    -   L represents a linker of formula (II):

wherein:

-   -   L1 is of formula (III):

-   -   wherein:        -   when P represents a hydrogen atom, then x=0 or 1 and y=1 and            z=0;        -   when P represents —OH, then x=y=z=0;        -   when P represents an activated O, then x=1 and y=z=0, or            x=y=z=1;        -   J₁, J₂, J₃ and J₄ are chosen, independently of each other,            from CA₁ and N;        -   ALK represents a (C₁-C₁₂)alkylene group, for instance            (C₁-C₆)alkylene, such as of the form —(CH₂)_(n)—, n being an            integer ranging from 1 to 12 and for example ranging from 1            to 6;        -   A₁, A₂, A₃, A₄, A₅, and A₆ represent, independently of each            other, a hydrogen atom or a (C₁-C₆)alkyl group, such as a            hydrogen atom or a methyl group.    -   (AA)w represents a sequence of w substituted AA_(s) or        non-substituted amino acids AA_(ns) connected together via        peptide bonds wherein substituted AA_(s) or non-substituted        AA_(ns) is defined below;    -   w represents an integer ranging from 1 to 12, for instance from        1 to 6, such as 2 or 3;

if (AA)w contains at least one substituted amino acid AA_(s), then L2represents a single bond, a (C₁-C₆)alkyl group, a(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—O(C₁-C₆)alkyl group, a(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, a CH(SO₃H)—(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-CH(SO₃H) group, a (C₁-C₆)alkyl-cyclohexyl group, aC(═O)—(C₁-C₆)alkyl group, a C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—O(C₁-C₆)alkyl group, aC(═O)—(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, a C(═O)—CH(SO₃H)—(C₁-C₆)alkylgroup, a C(═O)—(C₁-C₆)alkyl-CH(SO₃H) group, aC(═O)—(C₁-C₆)alkyl-cyclohexyl group, a NA₈-(C₁-C₆)alkyl group, aNA₈-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aNA₈-(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—O(C₁-C₆)alkyl group, aNA₈-(CH₂CH₂O)(C₁-C₆)alkyl group, a NA₈-(C₁-C₆)alkyl-CH(SO₃H) group, aC(═O)—NA₈-(C₁-C₆)alkyl group, a C(═O)—NA₈-(C₁-C₆)alkyl-(OCH₂CH₂)_(i)group, a C(═O)—NA₈-(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—O(C₁-C₆)alkyl group, aC(═O)—NA₈-(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-CH(SO₃H) group, a NA₇-(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-NA₇ group, a NA₇-(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, aNA₇-aryl group, a NA₇-heteroaryl group, a(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alkyl group, aC(═O)—NA₇-(C₁-C₆)alkyl group, a C(═O)—(C₁-C₆)alkyl-NA₇ group, aC(═O)—NA₇-(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, a C(═O)—NA₇-aryl group, aC(═O)—NA₇-heteroaryl group, a C(═O)—(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkylgroup, a C(═O)—(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, aC(═O)—(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, aC(═O)—(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alkyl group, aNA₈-(C₁-C₆)alkyl-NA₇ group, a NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkylgroup, a NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aNA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, aNA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aNA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, aNA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aNA₈-(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇ group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alky-(OCH₂CH₂)_(i) group, aC(═O)—NA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, aC(═O)—NA₆-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group or aC(═O)—NA₈-(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alkyl group;

if (AA)w represents a sequence of w non-substituted amino acids AA_(ns),L2 represents a NA₇-(C₁-C₆)alkyl group, a (C₁-C₆)alkyl-NA₇ group, aNA₇-(CH₂CH₂O)(C₁-C₆)alkyl group, a NA₇-aryl group, a NA₇-heteroarylgroup, a (C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alkyl group, aC(═O)—NA₇-(C₁-C₆)alkyl group, a C(═O)—(C₁-C₆)alkyl-NA₇ group, aC(═O)—NA₇-(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, a C(═O)—NA₇-aryl group, aC(═O)—NA₇-heteroaryl group, a C(═O)—(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkylgroup, a C(═O)—(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, aC(═O)—(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, aC(═O)—(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alkyl group, aNA₈-(C₁-C₆)alkyl-NA₇ group, a NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkylgroup, a NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aNA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, aNA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aNA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, aNA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aNA₈-(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇ group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl group, aC(═O)-NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—NA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group or aC(═O)—NA₈-(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alkyl group;

-   -   A₇ representing a straight or branched, saturated or        unsaturated, optionally substituted C₁-C₁₆₀ hydrocarbon chain        wherein optionally at least one methylene unit is independently        replaced by —NHC(═O)—, —N(alkyl)C(═O)—, —C(═O)NH—,        —C(═O)N(alkyl)-, —OC(═O)—, —C(═O)O—, —OC(═O)O—, —CH(OH)—,        —CH(SO₃H)—, —O—, —C(═O)—, —S(═O)—, —S(═O)₂—, —NHS(═O)₂—,        —N(alkyl)S(═O)₂—, —S(═O)₂NH—, —SO₂N(alkyl)-, —P(═O)(OH)—,        —P(═O)(OH)O—, —O—P(═O)(OH)—, —O—P(═O)(OH)—O— or a        heterocycloalkyl group optionally substituted with at least one        substituent, identical or different, chosen from —OH, —Oalkyl,        -alkyl, a halogen atom, —NH₂, —NHalkyl, and —N(alkyl)₂;    -    being understood that each A₇ comprising a SO₃H function can be        under salt forms such as alkali metal salts, for instance sodium        salts (SO₃ ⁻ ⁺Na);    -   A₈ representing a hydrogen atom or a (C₁-C₆)alkyl group, for        instance a hydrogen atom or a methyl group;    -   i representing an integer ranging from 1 to 50, for instance        ranging from 1 to 35.

The disclosure further relates to cryptophycin payloads of formula (IV):

wherein:

-   -   R₁ represents a (C₁-C₆)alkyl group;    -   R₂ and R₃ represent, independently of each other, a hydrogen        atom or a (C₁-C₆)alkyl group;    -    or alternatively R₂ and R₃ form together with the carbon atom        to which they are attached a (C₃-C₆)cycloalkyl or a        (C₃-C₆)heterocycloalkyl group;    -   R₄ and R₅ represent, independently of each other, a hydrogen        atom or a (C₁-C₆)alkyl group or a (C₁-C₆)alkyl-NH(R₁₂) group or        a (C₁-C₆)alkyl-OH group or a (C₁-C₆)alkyl-SH group or a        (C₁-C₆)alkyl-CO₂H group;    -    or alternatively R₄ and R₅ form together with the carbon atom        to which they are attached a (C₃-C₆)cycloalkyl or a        (C₃-C₆)heterocycloalkyl group;    -   X represents O or N(R₆);    -   R₆ represents a hydrogen atom or a (C₁-C₆)alkyl group;    -   R₇ and R₈ represent, independently of each other, a hydrogen        atom or a (C₁-C₆)alkyl group or a (C₁-C₆)alkyl-CO₂H group or a        (C₁-C₆)alkyl-N(C₁-C₆)alkyl₂ group; or alternatively R₇ and R₈        form together with the carbon atom to which they are attached a        (C₃-C₆)cycloalkyl group or a (C₃-C₆)heterocycloalkyl group;    -   R₉ represents at least one substituent of the phenyl nucleus        chosen, independently of each other, from: a hydrogen atom, —OH,        (C₁-C₄)alkoxy, a halogen atom, —NH₂, —NH(C₁-C₆)alkyl,        —N(C₁-C₆)alkyl₂, —NH(C₁-C₆)cycloalkyl or        (C₃-C₆)heterocycloalkyl;    -   R₁₀ represents at least one substituent of the phenyl nucleus        chosen from a hydrogen atom and a (C₁-C₄)alkyl group;    -   Y represents        -   —NR₁₁—(C₁-C₆)alkyl-, such as —NR₁₁—(CH₂)_(n)— like

-   -   -   —O—(C₁-C₆)alkyl-, such as —O—(CH₂)_(n)— like

-   -   -   —S—(C₁-C₆)alkyl-, such as —S—(CH₂)_(n)— like

-   -   Y being positioned in an ortho (o), meta (m) or para (p)        position of the phenyl nucleus;    -   R₁₁ and R₁₂ represent, independently of each other, a hydrogen        atom or (C₁-C₆)alkyl, such as a hydrogen atom or a methyl group;    -   n represents an integer ranging from 1 to 6.    -   L is defined as in formula (I) and represents a linker of        formula (II);    -   RCG1 represents a reactive chemical group present at the end of        the linker, RCG1 being reactive towards a chemical group present        on a polypeptide such as an antibody.

The present disclosure further relates to conjugates of formula (V):

wherein:

-   -   R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁ and R₁₂ are as        defined in formula (IV);    -   X, Y and L are as defined as in formula (IV);    -   G represents the product of reaction between RCG1, a reactive        chemical group present at the end of the linker and RCG2, an        orthogonal reactive chemical group present on the antibody (Ab);    -   Ab represents an antibody.

Each substituent R₁ to R₁₂ may also adopt one of the spatialconfigurations (e.g. R or S or alternatively Z or E) as described in theexamples.

The compounds of formula (I), (II), (III), (IV) and (V) may contain atleast one asymmetric carbon atoms. They may therefore exist in the formof enantiomers or diastereoisomers. These enantiomers anddiastereoisomers, and also mixtures thereof, including racemic mixtures,form part of this disclosure.

The compounds of formula (I), (II), (IV), including those that areillustrated, may exist in the form of bases or of acid-addition salts,for instance of pharmaceutically acceptable acids. For example, if thesecompounds comprise a SO₃H function, they may exist in the form of SO₃ ⁻alkali metal salts, such as of SO₃ ⁻ sodium salts (SO₃ ⁻ ⁺Na).

Definitions

In the context of the present disclosure, certain terms have thefollowing definitions:

-   -   alkenyl group: a hydrocarbon group obtained by removing one        hydrogen atom from an alkene. The alkenyl group may be linear or        branched. Examples that may be mentioned include ethenyl        (—CH═CH₂, also named vinyl) and propenyl (—CH₂—CH═CH₂, also        named allyl).    -   alkoxy group: a group —O-alkyl, in which the alkyl group is as        defined below;    -   alkyl group: a linear or branched saturated aliphatic        hydrocarbon-based group obtained by removing a hydrogen atom        from an alkane. Examples that may be mentioned include methyl,        ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl,        neopentyl, 2,2-dimethylpropyl and hexyl groups;    -   alkylene group: a saturated divalent group of empirical formula        —C_(n)H_(2n)—, obtained by removing two hydrogen atoms from an        alkane. The alkylene group may be linear or branched. Examples        that may be mentioned include methylene (—CH₂—), ethylene        (—CH₂CH₂—), propylene (—CH₂CH₂CH₂—), butylene (—CH₂CH₂CH₂CH₂—)        and hexylene (—CH₂CH₂CH₂CH₂CH₂CH₂—) groups or the following        branched groups

for instance, the alkylene group is of the formula —(CH₂)_(n)—, nrepresenting an integer; in the ranges of values, the limits areincluded (e.g. a range of the type “n ranging from 1 to 6” or “rangingfrom 1 to 6” includes limits 1 and 6);

-   -   antibody: an antibody with affinity for a biological target,        such as a monoclonal antibody. The function of the antibody is        to direct the biologically active compound as a cytotoxic        compound towards the biological target. The antibody may be        monoclonal, polyclonal or multispecific; it may also be an        antibody fragment; it may also be a murine, chimeric, humanized        or human antibody. An “antibody” may be a natural or        conventional antibody in which two heavy chains are linked to        each other by disulfide bonds and each heavy chain is linked to        a light chain by a disulfide bond (also referred to as a        “full-length antibody”). The terms “conventional (or        full-length) antibody” refers both to an antibody comprising the        signal peptide (or pro-peptide, if any), and to the mature form        obtained upon secretion and proteolytic processing of the        chain(s). There are two types of light chain, lambda (I) and        kappa (k). There are five main heavy chain classes (or isotypes)        which determine the functional activity of an antibody molecule:        IgM, IgD, IgG, IgA and IgE. Each chain contains distinct        sequence domains. The light chain includes two domains or        regions, a variable domain (VL) and a constant domain (CL). The        heavy chain includes four domains, a variable domain (VH) and        three constant domains (CH1, CH2 and CH3, collectively referred        to as CH). The variable regions of both light (VL) and heavy        (VH) chains determine binding recognition and specificity to the        antigen. The constant region domains of the light (CL) and heavy        (CH) chains confer important biological properties such as        antibody chain association, secretion, trans-placental mobility,        complement binding, and binding to Fc receptors (FcR). The Fv        fragment is the N-terminal part of the Fab fragment of an        immunoglobulin and consists of the variable portions of one        light chain and one heavy chain. The specificity of the antibody        resides in the structural complementarity between the antibody        combining site and the antigenic determinant. Antibody combining        sites are made up of residues that are primarily from the        hypervariable or complementarity determining regions (CDRs).        Occasionally, residues from nonhypervariable or framework        regions (FR) influence the overall domain structure and hence        the combining site. CDRs refer to amino acid sequences which        together define the binding affinity and specificity of the        natural Fv region of a native immunoglobulin binding site. The        light and heavy chains of an immunoglobulin each have three        CDRs, designated CDR1-L, CDR2-L, CDR3-L and CDR1-H, CDR2-H,        CDR3-H, respectively. A conventional antibody antigen-binding        site, therefore, includes six CDRs, comprising the CDR set from        each of a heavy and a light chain V region.

As used herein, the term “antibody” denotes both conventional(full-length) antibodies and fragments thereof, as well as single domainantibodies and fragments thereof, such as variable heavy chain of singledomain antibodies. Fragments of (conventional) antibodies typicallycomprise a portion of an intact antibody, such as the antigen bindingregion or variable region of the intact antibody, and retain thebiological function of the conventional antibody. Examples of suchfragments include Fv, Fab, F(ab′)2, Fab′, dsFv, (dsFv)2, scFv, sc(Fv)2,nanobodies and diabodies.

The function of the antibody is to direct the biologically activecompound as a cytotoxic compound towards the biological target.

-   -   aryl group: a cyclic aromatic group containing ranging from 5 to        10 carbon atoms. Examples of aryl groups include phenyl, tolyl,        xylyl, naphthyl;    -   biological target: an antigen (or group of antigens) for        instance located at the surface of cancer cells or stromal cells        associated with this tumour; these antigens may be, for example,        a growth factor receptor, an oncogene product or a mutated        “tumor suppressant” gene product, an angiogenesis-related        molecule or an adhesion molecule;    -   conjugate: an antibody-drug conjugate or ADC, i.e. a polypeptide        such as an antibody to which is covalently attached via a linker        at least one molecule of a cytotoxic compound;    -   cycloalkyl group: a cyclic alkyl group comprising carbon atoms        ranging from 3 and 6 engaged in the cyclic structure. Examples        that may be mentioned include cyclopropyl, cyclobutyl,        cyclopentyl and cyclohexyl groups;    -   DAR (drug-to-antibody ratio): an average number of cytotoxic        molecules attached via a linker to an antibody;    -   halogen: any of the four elements fluorine, chlorine, bromine        and iodine;    -   heteroaryl group: an aryl group containing carbon atoms ranging        from 2 to 10 and heteroatoms ranging from 1 to 5 such as        nitrogen, oxygen or sulphur engaged in the ring and connected to        the carbon atoms forming the ring. Examples of heteroaryl groups        include pyridyl, pyrimidyl, thienyl, imidazolyl, triazolyl,        indolyl, imidazo-pyridyl, pyrazolyl;    -   heterocycloalkyl group: a cycloalkyl group containing carbon        atoms ranging from 2 to 8 and heteroatoms ranging from 1 to 3,        such as nitrogen, oxygen or sulphur engaged in the ring and        connected to the carbon atoms forming the ring. Examples include        aziridinyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl,        thiomorpholinyl, tetrahydrofuranyl, tetrahydrothiofuranyl,        tetrahydropyranyl, azetidinyl, oxetanyl and pyranyl;    -   linker: a group of atoms or a single bond that can covalently        attach a cytotoxic compound to a polypeptide such as an antibody        in order to form a conjugate;    -   payload: a cytotoxic compound to which is covalently attached a        linker;    -   reactive chemical group: a group of atoms that can promote or        undergo a chemical reaction;    -   Further, as stated throughout the present disclosure, a SO₃H        function can be under salt forms such as alkali metal salts, for        instance sodium salts (SO₃ ⁻ ⁺Na).

Abbreviations

ADC: antibody-drug conjugate; ALK: (C₁-C₁₂)alkylene group, for instance(C₁-C₆)alkylene, such as of the form —(CH₂)_(n)—, n being an integerranging from 1 to 12 and for example ranging from 1 to 6; aq.: aqueous;Ar: argon; AUC: area under the curve; BCN:(1α,8α,9β)-bicyclo[6.1.0]non-4-yne-9-methanol; CHCl₃: chloroform; CH₃CN:acetonitrile; CO₂: carbon dioxide; CR: complete response; crypto denotesthe unit of formula

crypto for instance denoting a cryptophycin compound of an example, oneof the D₁-D₈ cryptophycin compounds described in WO2011/001052 or one ofthe D₁-D₁₉ cryptophycin compounds below as described inPCT/EP2016/076603:

-   -   wherein W represents        -   (C₁-C₆)alkyl-NH(R₁₁), such as (CH₂)_(n)NHR₁₁;        -   (C₁-C₆)alkyl-OH, such as (CH₂)_(n)OH;        -   (C₁-C₆)alkyl-SH, such as (CH₂)_(n)SH;        -   CO₂H or C(═O)NH₂;        -   (C₁-C₆)alkyl-CO₂H or (C₁-C₆)alkyl-C(═O)NH₂; or        -   (C₁-C₆)alkyl-N₃.

W is positioned in an ortho (o), meta (m) or para (p) position of thephenyl nucleus;

-   -   R₁₁ represents, independently of each other, a hydrogen atom or        a group (C₁-C₆)alkyl, for instance a hydrogen atom or a methyl        group;    -   n represents an integer ranging from 1 to 6;        d: day; DAR: drug-to-antibody ratio (D1 refers to an ADC with a        DAR of 1, D2 to an ADC with a DAR of 2, etc.); DBCO:        dibenzylcyclooctyne; DCC: N,N′-dicyclohexylcarbodiimide; DCM:        dichloromethane; DIEA: N,N-diisopropylethylamine; DMA:        dimethylacetamide; DMAP: 4-(dimethylamino)pyridine; DMEM:        Dulbecco's Modified Eagle Medium; DMEM/F12: Dulbecco's Modified        Eagle Medium Nutrient Mixture F-12; DMF: dimethylformamide;        DMSO: dimethylsulfoxide; DPBS: Dulbecco's phosphate-buffered        saline; DSC: N,N′-disuccinimidyl carbonate; EDC:        1-(3-dimethylaminopropyl)-3-ethylcarbodiimide; EDTA:        ethylenediaminetetraacetic acid; EEDQ:        N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline; ELSD:        evaporating light scattering detector; eq.: equivalent; ES:        electrospray; EtOAc: ethyl acetate; Et₂O: diethyl ether; Ex.:        example; FCS: foetal calf serum; Fmoc:        9-fluorenylmethoxycarbonyl; GI: electroinductive group; h: hour;        H₂O: water; Hal: halogen; HATU:        1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium        3-oxid hexafluorophosphate; HCl: chlorohydric acid; HEPES:        4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HIC:        hydrophobic interaction chromatography; HOAt:        1-hydroxy-7-azabenzotriazole; HOBt: 1-hydroxy-benzotriazole;        HPLC: high performance liquid chromtography; HRMS: high        resolution mass spectrometry; IC₅₀: median inhibitory        concentration; i.e.: id est meaning that is; IEC: ion exchange        chromatography; iPrOH: 2-propanol; iPr₂O: diisopropyl ether;        i.v.: intravenous; MeCN: acetonitrile; MeOH: methanol; MeTHF:        2-methyl-tetrahydrofuran; MFCO:        1-fluoro-2-cyclooctyne-1-carboxylic acid; MgSO₄: magnesium        sulfate; min: minute; MsCI: methanesulfonyl chloride; MTBE:        methyl tert-butyl ether; MTD: maximum tolerated dose; NaH:        sodium hydride; NaCl: sodium chloride; NaHCO₃: sodium hydrogen        carbonate; n.d.: not determined; NHS: N-hydroxysuccinimide; NMP:        1-methyl-2-pyrrolidone; NMR: nuclear magnetic resonance; PABA:        para-aminobenzyl alcohol; PBS: phosphate-buffered saline; PEG:        polyethylene glycol; PNGase F: Peptide-N-Glycosidase F; ppm:        parts per million; PR: partial response; QS: quantum satis        meaning the amount what is needed; Q-Tof: quadrupole        time-of-flight; quant.: quantitative yield; RCG: reactive        chemical group; RT: room temperature; sat.: saturated; s.c.:        subcutaneously; SCID: severe combined immunodeficiency; SEC:        size exclusion chromatography; T3P: propylphosphonic anhydride;        TBAF: tetrabutylammonium fluoride; TFA: trifluoroacetic acid;        TFS: tumor-free survivor; THF: tetrahydrofuran; TLC: thin layer        chromatography; t_(1/2): half-life; t_(R): retention time; UPLC:        Ultra Performance Liquid Chromatography; UV: ultra-violet.

FIGURES

FIG. 1: In vivo efficacy of Ex. 6 against MDA-MB-231 xenograft in SCIDmice

FIG. 2: In vivo efficacy of Ex. 16, Ex. 19, Ex. 23 and Ex. 32 againstMDA-MB-231 xenograft in SCID mice at 2.5 mg/kg

FIG. 3: In vivo efficacy of Ex. 16, Ex. 19, Ex. 23 and Ex. 32 againstMDA-MB-231 xenograft in SCID mice at 1.25 mg/kg

FIG. 4: In vivo efficacy of Ex. 26 against MDA-MB-231 xenograft in SCIDmice

FIG. 5: In vivo efficacy of Ex. 29 against MDA-MB-231 xenograft in SCIDmice

FIG. 6: In vivo efficacy of Ex. 35 against MDA-MB-231 xenograft in SCIDmice

FIG. 7: In vivo efficacy of Ex. 41 against MDA-MB-231 xenograft in SCIDmice

DETAILED DESCRIPTION OF THE PRESENT DISCLOSURE

The disclosure relates to new peptidic compounds, chosen from compoundsof formula (I):

RCG1-L-P  (I)

wherein

-   -   RCG1 represents a reactive chemical group that is reactive        towards a chemical group present on a polypeptide such as an        antibody;    -   L represents a linker of formula (II) as defined below;    -   P represents a hydrogen atom, —OH or an activated O.

Examples of activated O that may be mentioned include

where cation represents for example sodium, potassium or cesium;or,

group in which GI represents at least one electroinductive group such as—NO₂ or a halogen atom (-Hal), for instance a fluorine atom (—F). Theymay be, for example, the following groups:

The linker L which is present in formula (I) is of formula (II):

wherein:

-   -   L1 is of formula (III):

-   -   wherein:        -   when P represents a hydrogen atom, then x=0 or 1 and y=1 and            z=0;        -   when P represents —OH, then x=y=z=0;        -   when P represents an activated O, then x=1 and y=z=0, or            x=y=z=1;        -   J₁, J₂, J₃ and J₄ are chosen, independently of each other,            from CA₁ and N;        -   ALK represents a (C₁-C₁₂)alkylene group, for instance            (C₁-C₆)alkylene, such as of the form —(CH₂)_(n)—, n being an            integer ranging from 1 to 12 and for example ranging from 1            to 6;        -   A₁, A₂, A₃, A₄, A₅, and A₆ represent, independently of each            other, a hydrogen atom or a (C₁-C₆)alkyl group, such as a            hydrogen atom or a methyl group.    -   (AA)w represents a sequence of w substituted AA_(s) or        non-substituted amino acids AA_(ns) connected together via        peptide bonds wherein substituted AA_(s) or non-substituted        AA_(ns) is defined below;    -   w represents an integer ranging from 1 to 12, for instance from        1 to 6, such as 2 or 3;

if (AA)w contains at least one substituted amino acid AA_(s), then L2represents:

a single bond, a (C₁-C₆)alkyl group, a (C₁-C₆)alkyl-(OCH₂CH₂)_(i) group,a (C₁-C₆)alkyl-(OCH₂CH₂)_(i)—O(C₁-C₆)alkyl group, a(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, a CH(SO₃H)—(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-CH(SO₃H) group, a (C₁-C₆)alkyl-cyclohexyl group, aC(═O)—(C₁-C₆)alkyl group, a C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—O(C₁-C₆)alkyl group, aC(═O)—(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, a C(═O)—CH(SO₃H)—(C₁-C₆)alkylgroup, a C(═O)—(C₁-C₆)alkyl-CH(SO₃H) group, aC(═O)—(C₁-C₆)alkyl-cyclohexyl group, a NA₈-(C₁-C₆)alkyl group, aNA₈-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aNA₈-(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—O(C₁-C₆)alkyl group, aNA₈-(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, a NA₈-(C₁-C₆)alkyl-CH(SO₃H) group,a C(═O)—NA₈-(C₁-C₆)alkyl group, a C(═O)—NA₈-(C₁-C₆)alkyl-(OCH₂CH₂)_(i)group, a C(═O)—NA₈-(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—O(C₁-C₆)alkyl group, aC(═O)—NA₈-(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-CH(SO₃H) group, a NA₇-(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-NA₇ group, a NA₇-(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, aNA₇-aryl group, a NA₇-heteroaryl group, a(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alkyl group, aC(═O)—NA₇-(C₁-C₆)alkyl group, a C(═O)—(C₁-C₆)alkyl-NA₇ group, aC(═O)—NA₇-(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, a C(═O)—NA₇-aryl group, aC(═O)—NA₇-heteroaryl group, a C(═O)—(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkylgroup, a C(═O)—(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, aC(═O)—(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, aC(═O)—(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alkyl group, aNA₈-(C₁-C₆)alkyl-NA₇ group, a NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkylgroup, a NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aNA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, aNA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aNA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, aNA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aNA₈-(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alky group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇ group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alky-(OCH₂CH₂)_(i) group, aC(═O)—NA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group or aC(═O)—NA₈-(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alkyl group;

if (AA)w represents a sequence of w non-substituted amino acids AA_(ns),then L2 represents:

a NA₇-(C₁-C₆)alkyl group, a (C₁-C₆)alkyl-NA₇ group, aNA₇-(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, a NA₇-aryl group, a NA₇-heteroarylgroup, a (C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alkyl group, aC(═O)—NA₇-(C₁-C₆)alkyl group, a C(═O)—(C₁-C₆)alkyl-NA₇ group, aC(═O)—NA₇-(CH₂CH₂O)(C₁-C₆)alkyl group, a C(═O)—NA₇-aryl group, aC(═O)—NA₇-heteroaryl group, a C(═O)—(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkylgroup, a C(═O)—(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, aC(═O)—(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, aC(═O)—(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alkyl group, aNA₈-(C₁-C₆)alkyl-NA₇ group, a NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkylgroup, a NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aNA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, aNA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aNA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, aNA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aNA₈-(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alky group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇ group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—NA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group or aC(═O)—NA₈-(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alkyl group;

-   -   A₇ representing a straight or branched, saturated or        unsaturated, optionally substituted C₁-C₁₆₀ hydrocarbon chain        wherein optionally at least one methylene unit is independently        replaced by —NHCO—, —N(alkyl)C(═O)—, —C(═O)NH—, —C(═O)N(alkyl)-,        —OC(═O)—, —C(═O)O—, —OC(═O)O—, —CH(OH)—, —CH(SO₃H)—, —O—,        —C(═O)—, —S(═O)—, —S(═O)₂—, —NHS(═O)₂—, —N(alkyl)S(═O)₂—,        —S(═O)₂NH—, —S(═O)₂N(alkyl)-, —P(═O)(OH)—, —P(═O)(OH)O—,        —O—P(═O)(OH)—, —O—P(═O)(OH)—O— or a heterocycloalkyl group        optionally substituted with at least one substituent, identical        or different, chosen from —OH, —Oalkyl, -alkyl, a halogen atom,        —NH₂, —NHalkyl, and —N(alkyl)₂; being understood that each A₇        comprising a SO₃H function can be under salt forms such as        alkali metal salts, for instance sodium salts (SO₃ ⁻ ⁺Na).    -   A₈ representing a hydrogen atom or a (C₁-C₆)alkyl group, for        instance a hydrogen atom or a methyl group;    -   i representing an integer ranging from 1 to 50, such as ranging        from 1 to 35.

According to a particular embodiment, the disclosure relates tocompounds of formula (I):

RCG1-L-P  (I)

wherein

-   -   RCG1 represents a reactive chemical group that is reactive        towards a chemical group present on a polypeptide such as an        antibody;    -   P represents a hydrogen atom, —OH or an activated O;    -   L represents a linker of formula (II):

wherein:

-   -   L1 is of formula (III):

-   -   wherein:        -   when P represents a hydrogen atom, then x=0 or 1 and y=1 and            z=0;        -   when P represents —OH, then x=y=z=0;        -   when P represents an activated O, then x=1 and y=z=0, or            x=y=z=1;        -   J₁, J₂, J₃ and J₄ are chosen, independently of each other,            from CA₁ and N;        -   ALK represents a (C₁-C₁₂)alkylene group, for instance            (C₁-C₆)alkylene, such as of the form —(CH₂)_(n)—, n being an            integer ranging from 1 to 12 and for example ranging from 1            to 6;        -   A₁, A₂, A₃, A₄, A₅, and A₆ represent, independently of each            other, a hydrogen atom or a (C₁-C₆)alkyl group, such as a            hydrogen atom or a methyl group.    -   (AA)w represents a sequence of w substituted AA_(s) or        non-substituted amino acids AA_(ns) connected together via        peptide bonds;    -   w represents an integer ranging from 1 to 12, for instance from        1 to 6, such as 2 or 3;

if (AA)w contains at least one substituted amino acid AA_(s), then L2represents a single bond, a (C₁-C₆)alkyl group, a(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—O(C₁-C₆)alkyl group, a(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, a CH(SO₃H)—(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-CH(SO₃H) group, a (C₁-C₆)alkyl-cyclohexyl group, aC(═O)—(C₁-C₆)alkyl group, a C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—O(C₁-C₆)alkyl group, aC(═O)—(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, a C(═O)—CH(SO₃H)—(C₁-C₆)alkylgroup, a C(═O)—(C₁-C₆)alkyl-CH(SO₃H) group, aC(═O)—(C₁-C₆)alkyl-cyclohexyl group, a NA₈-(C₁-C₆)alkyl group, aNA₈-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aNA₈-(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—O(C₁-C₆)alkyl group, aNA₈-(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, a NA₈-(C₁-C₆)alkyl-CH(SO₃H) group,a C(═O)—NA₈-(C₁-C₆)alkyl group, a C(═O)—NA₈-(C₁-C₆)alkyl-(OCH₂CH₂)_(i)group, a C(═O)—NA₈-(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—O(C₁-C₆)alkyl group, aC(═O)—NA₈-(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-CH(SO₃H) group, a NA₇-(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-NA₇ group, a NA₇-(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, aNA₇-aryl group, a NA₇-heteroaryl group, a(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alkyl group, aC(═O)—NA₇-(C₁-C₆)alkyl group, a C(═O)—(C₁-C₆)alkyl-NA₇ group, aC(═O)—NA₇-(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, a C(═O)—NA₇-aryl group, aC(═O)—NA₇-heteroaryl group, a C(═O)—(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkylgroup, a C(═O)—(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, aC(═O)—(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, aC(═O)—(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alkyl group, aNA₈-(C₁-C₆)alkyl-NA₇ group, a NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkylgroup, a NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aNA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, aNA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aNA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, aNA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aNA₈-(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇ group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alky-(OCH₂CH₂)_(i) group, aC(═O)—NA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group or aC(═O)—NA₈-(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alkyl group;

if (AA)w represents a sequence of w non-substituted amino acids AA_(ns),then L2 represents a NA₇-(C₁-C₆)alkyl group, a (C₁-C₆)alkyl-NA₇ group, aNA₇-(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, a NA₇-aryl group, a NA₇-heteroarylgroup, a (C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alkyl group, aC(═O)—NA₇-(C₁-C₆)alkyl group, a C(═O)—(C₁-C₆)alkyl-NA₇ group, aC(═O)—NA₇-(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, a C(═O)—NA₇-aryl group, aC(═O)—NA₇-heteroaryl group, a C(═O)—(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkylgroup, a C(═O)—(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, aC(═O)—(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, aC(═O)—(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alkyl group, aNA₈-(C₁-C₆)alkyl-NA₇ group, a NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkylgroup, a NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aNA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, aNA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aNA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, aNA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aNA₈-(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alky group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇ group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—NA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group or aC(═O)—NA₈-(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alkyl group;

-   -   A₇ representing a straight or branched, saturated or        unsaturated, optionally substituted C₁-C₁₆₀ hydrocarbon chain        wherein optionally at least one methylene unit is independently        replaced by —NHC(═O)—, —N(alkyl)C(═O)—, —C(═O)NH—,        —C(═O)N(alkyl)-, —OC(═O)—, —C(═O)O—, —OC(═O)O—, —CH(OH)—,        —CH(SO₃H)—, —O—, —C(═O)—, —S(═O)—, —S(═O)₂—, —NHS(═O)₂—,        —N(alkyl)S(═O)₂—, —S(═O)₂NH—, —S(═O)₂N(alkyl)-, —P(═O)(OH)—,        —P(═O)(OH)O—, —O—P(═O)(OH)—, —O—P(═O)(OH)—O— or a        heterocycloalkyl group optionally substituted with at least one        substituent, identical or different, chosen from —OH, —Oalkyl,        -alkyl, a halogen atom, —NH₂, —NHalkyl, and —N(alkyl)₂, being        understood that each A₇ comprising a SO₃H function can be under        salt forms such as alkali metal salts, for instance sodium salts        (SO₃ ⁻ ⁺Na);    -   A₈ representing a hydrogen atom or a (C₁-C₆)alkyl group, for        instance a hydrogen atom or a methyl group;    -   i representing an integer ranging from 1 to 50, for instance        ranging from 1 to 35,    -   a non-substituted amino acid AA_(ns) denoting natural or        non-natural amino acid, of configuration D or L, identical to or        derived from: alanine (Ala), β-alanine, γ-aminobutyric acid,        2-amino-2-cyclohexylacetic acid, 2-amino-2-phenylacetic acid,        arginine (Arg), asparagine (Asn), aspartic acid (Asp),        citrulline (Cit), cysteine (Cys), α,α-dimethyl-γ-aminobutyric        acid, β,β-dimethyl-γ-aminobutyric acid, glutamine (Gln),        glutamic acid (Glu), glycine (Gly), homo-cysteine,        selenocysteine, homo-selenocysteine, histidine (His), isoleucine        (Ile), leucine (Leu), lysine (Lys), ε-acetyl-lysine (AcLys),        methionine (Met), ornithine (Orn), phenylalanine (Phe), proline        (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine        (Tyr), and valine (Val).

According to another particular embodiment, the disclosure relates tocompounds of formula (I):

RCG1-L-P  (I)

wherein

-   -   RCG1 represents a reactive chemical group that is reactive        towards a chemical group present on a polypeptide such as an        antibody;    -   P represents a hydrogen atom, —OH or an activated O;    -   L represents a linker of formula (II):

wherein:

-   -   L1 is of formula (III):

-   -   wherein:        -   when P represents a hydrogen atom, then x=0 or 1 and y=1 and            z=0;        -   when P represents —OH, then x=y=z=0;        -   when P represents an activated O, then x=1 and y=z=0, or            x=y=z=1;        -   J₁, J₂, J₃ and J₄ are chosen, independently of each other,            from CA₁ and N;        -   ALK represents a (C₁-C₁₂)alkylene group, for instance            (C₁-C₆)alkylene, such as of the form —(CH₂)_(n)—, n being an            integer ranging from 1 to 12 and for example ranging from 1            to 6;        -   A₁, A₂, A₃, A₄, A₅, and A₆ represent, independently of each            other, a hydrogen atom or a (C₁-C₆)alkyl group, such as a            hydrogen atom or a methyl group.    -   (AA)w represents a sequence of w substituted AA_(s) or        non-substituted amino acids AA_(ns) connected together via        peptide bonds;    -   w represents an integer ranging from 1 to 12, for instance from        1 to 6, such as 2 or 3;

if (AA)w contains at least one substituted amino acid AA_(s), then L2represents a single bond, a (C₁-C₆)alkyl group, a(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—O(C₁-C₆)alkyl group, a(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, a CH(SO₃H)—(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-CH(SO₃H) group, a (C₁-C₆)alkyl-cyclohexyl group, aC(═O)—(C₁-C₆)alkyl group, a C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—O(C₁-C₆)alkyl group, aC(═O)—(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, a C(═O)—CH(SO₃H)—(C₁-C₆)alkylgroup, a C(═O)—(C₁-C₆)alkyl-CH(SO₃H) group, aC(═O)—(C₁-C₆)alkyl-cyclohexyl group, a NA₈-(C₁-C₆)alkyl group, aNA₈-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aNA₈-(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—O(C₁-C₆)alkyl group, aNA₈-(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, a NA₈-(C₁-C₆)alkyl-CH(SO₃H) group,a C(═O)—NA₈-(C₁-C₆)alkyl group, a C(═O)—NA₈-(C₁-C₆)alkyl-(OCH₂CH₂)_(i)group, a C(═O)—NA₈-(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—O(C₁-C₆)alkyl group, aC(═O)—NA₈-(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-CH(SO₃H) group, a NA₇-(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-NA₇ group, a NA₇-(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, aNA₇-aryl group, a NA₇-heteroaryl group, a(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alkyl group, aC(═O)—NA₇-(C₁-C₆)alkyl group, a C(═O)—(C₁-C₆)alkyl-NA₇ group, aC(═O)—NA₇-(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, a C(═O)—NA₇-aryl group, aC(═O)—NA₇-heteroaryl group, a C(═O)—(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkylgroup, a C(═O)—(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, aC(═O)—(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, aC(═O)—(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alkyl group, aNA₈-(C₁-C₆)alkyl-NA₇ group, a NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkylgroup, a NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aNA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, aNA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aNA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, aNA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aNA₈-(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alky group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇ group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alky-(OCH₂CH₂)_(i) group, aC(═O)—NA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group or aC(═O)—NA₈-(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alkyl group;

if (AA)w represents a sequence of w non-substituted amino acids AA_(ns),then L2 represents a NA₇-(C₁-C₆)alkyl group, a (C₁-C₆)alkyl-NA₇ group, aNA₇-(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, a NA₇-aryl group, a NA₇-heteroarylgroup, a (C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alkyl group, aC(═O)—NA₇-(C₁-C₆)alkyl group, a C(═O)—(C₁-C₆)alkyl-NA₇ group, aC(═O)—NA₇-(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, a C(═O)—NA₇-aryl group, aC(═O)—NA₇-heteroaryl group, a C(═O)—(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkylgroup, a C(═O)—(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, aC(═O)—(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, aC(═O)—(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alkyl group, aNA₈-(C₁-C₆)alkyl-NA₇ group, a NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkylgroup, a NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aNA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, aNA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aNA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, aNA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aNA₈-(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇ group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—NA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group or aC(═O)—NA₆-(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alkyl group;

-   -   A₇ representing a straight or branched, saturated or        unsaturated, optionally substituted C₁-C₁₆₀ hydrocarbon chain        wherein optionally at least one methylene unit is independently        replaced by —NHC(═O)—, —N(alkyl)C(═O)—, —C(═O)NH—,        —C(═O)N(alkyl)-, —OC(═O)—, —C(═O)O—, —OC(═O)O—, —CH(OH)—,        —CH(SO₃H)—, —O—, —C(═O)—, —S(═O)—, —S(═O)₂—, —NHS(═O)₂—,        —N(alkyl)S(═O)₂—, —S(═O)₂NH—, —S(═O)₂N(alkyl)-, —P(═O)(OH)—,        —P(═O)(OH)O—, —O—P(═O)(OH)—, —O—P(═O)(OH)—O— or a        heterocycloalkyl group optionally substituted with at least one        substituent, identical or different, chosen from —OH, —Oalkyl,        -alkyl, a halogen atom, —NH₂, —NHalkyl, and —N(alkyl)₂, being        understood that each A₇ comprising a SO₃H function can be under        salt forms such as alkali metal salts, for instance sodium salts        (SO₃ ⁻ ⁺Na);    -   A₈ representing a hydrogen atom or a (C₁-C₆)alkyl group, for        instance a hydrogen atom or a methyl group;    -   i representing an integer ranging from 1 to 50, for instance        ranging from 1 to 35, wherein said substituted amino acids        AA_(s) have the formula (VI):

wherein:

-   -   T represents a saturated or unsaturated, linear or branched,        (C₁-C₈) trivalent alkyl group, preferably T is

-   -   U group represents a single bond, —NHC(═O)—, —N(alkyl)C(═O)—,        —C(═O)NH—, —C(═O)N(alkyl)-, —NHC(═O)NH—, —NHC(═NH)NH—, —OC(═O)—,        —C(═O)O—, —OC(═O)O—, —S—, —Se—, —O—, —NH—, —N(alkyl)-, —C(═O)—,        —OP(═O)—, —S(═O)—, —S(═O)₂—, —NHS(═O)₂—, —N(alkyl)S(═O)₂—,        —S(═O)₂NH—, —S(═O)₂N(alkyl)-, —P(═O)(OH)—, —P(═O)(OH)O—,        —O—P(═O)(OH)—, or —O—P(═O)(OH)—O, such as U group represents        —NH—C(═O)—, or —C(═O)NH—;    -   A₉ represents a straight or branched, saturated or unsaturated,        optionally substituted C₁-C₁₆₀ hydrocarbon chain wherein        optionally at least one methylene unit is independently replaced        by —NH—C(═O)—, —N(alkyl)C(═O)—, —C(═O)NH—, —C(═O)N(alkyl)-,        —NHC(═O)NH—, —NHC(═NH)NH—, —OC(═O)—, —C(═O)O—, —OC(═O)O—,        —CH(OH)—, —CH(SO₃H)—, —CH(Oalkyl)-, —CHF—, —CF₂—, —S—, —Se—,        —O—, —NH—, —N(alkyl)-, —N⁺H(alkyl)-, —N⁺(alkyl)₂-, —C(═O)—,        —OP(═O)—, —S(═O)—, —S(═O)₂—, —NHS(═O)₂—, —N(alkyl)S(═O)₂—,        —S(═O)₂NH—, —S(═O)₂N(alkyl)-, —P(═O)(OH)—, —P(═O)(OH)O—,        —O—P(═O)(OH)—, —O—P(═O)(OH)—O— or a heterocycloalkyl group        optionally substituted with at least one substituent, identical        or different, chosen from —OH, —Oalkyl, -alkyl, a halogen atom,        —NH₂, —NHalkyl, and —N(alkyl)₂, for instance A₉ is        —[(CH₂)₂—O]₄—CH₃, —[(CH₂)₂—O]₂₄—CH₃,

or —[(CH₂)₂—O)]₄—(CH₂)₂—C(═O)—NH—(CH₂)₂—SO₃H;being understood that each A₉ comprising a SO₃H function can be undersalt forms such as alkali metal salts, for instance sodium salts (SO₃ ⁻⁺Na).

According to another particular embodiment, the disclosure relates tocompounds of formula (I):

RCG1-L-P  (I)

wherein

-   -   RCG1 represents a reactive chemical group that is reactive        towards a chemical group present on a polypeptide such as an        antibody;    -   P represents a hydrogen atom, —OH or an activated O;    -   L represents a linker of formula (II):

wherein:

-   -   L1 is of formula (III):

-   -   wherein:        -   when P represents a hydrogen atom, then x=0 or 1 and y=1 and            z=0;        -   when P represents —OH, then x=y=z=0;        -   when P represents an activated O, then x=1 and y=z=0, or            x=y=z=1;        -   J₁, J₂, J₃ and J₄ are chosen, independently of each other,            from CA₁ and N;        -   ALK represents a (C₁-C₁₂)alkylene group, for instance            (C₁-C₆)alkylene, such as of the form —(CH₂)_(n)—, n being an            integer ranging from 1 to 12 and for example ranging from 1            to 6;        -   A₁, A₂, A₃, A₄, A₅, and A₆ represent, independently of each            other, a hydrogen atom or a (C₁-C₆)alkyl group, such as a            hydrogen atom or a methyl group.    -   (AA)w represents a sequence of w substituted AA_(s) or        non-substituted amino acids AA_(ns) connected together via        peptide bonds;    -   w represents an integer ranging from 1 to 12, for instance from        1 to 6, such as 2 or 3;

if (AA)w contains at least one substituted amino acid AA_(s), then L2represents a single bond, a (C₁-C₆)alkyl group, a(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—O(C₁-C₆)alkyl group, a(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, a CH(SO₃H)—(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-CH(SO₃H) group, a (C₁-C₆)alkyl-cyclohexyl group, aC(═O)—(C₁-C₆)alkyl group, a C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—O(C₁-C₆)alkyl group, aC(═O)—(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, a C(═O)—CH(SO₃H)—(C₁-C₆)alkylgroup, a C(═O)—(C₁-C₆)alkyl-CH(SO₃H) group, aC(═O)—(C₁-C₆)alkyl-cyclohexyl group, a NA₈-(C₁-C₆)alkyl group, aNA₈-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aNA₈-(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—O(C₁-C₆)alkyl group, aNA₈-(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, a NA₈-(C₁-C₆)alkyl-CH(SO₃H) group,a C(═O)—NA₈-(C₁-C₆)alkyl group, a C(═O)—NA₈-(C₁-C₆)alkyl-(OCH₂CH₂)_(i)group, a C(═O)—NA₈-(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—O(C₁-C₆)alkyl group, aC(═O)—NA₈-(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-CH(SO₃H) group, a NA₇-(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-NA₇ group, a NA₇-(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, aNA₇-aryl group, a NA₇-heteroaryl group, a(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alkyl group, aC(═O)—NA₇-(C₁-C₆)alkyl group, a C(═O)—(C₁-C₆)alkyl-NA₇ group, aC(═O)—NA₇-(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, a C(═O)—NA₇-aryl group, aC(═O)—NA₇-heteroaryl group, a C(═O)—(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkylgroup, a C(═O)—(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, aC(═O)—(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, aC(═O)—(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alkyl group, aNA₈-(C₁-C₆)alkyl-NA₇ group, a NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkylgroup, a NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aNA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, aNA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aNA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, aNA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aNA₈-(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇ group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alky-(OCH₂CH₂)_(i) group, aC(═O)—NA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group or aC(═O)—NA₈-(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alkyl group;

if (AA)w represents a sequence of w non-substituted amino acids AA_(ns),then L2 represents a NA₇-(C₁-C₆)alkyl group, a (C₁-C₆)alkyl-NA₇ group, aNA₇-(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, a NA₇-aryl group, a NA₇-heteroarylgroup, a (C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alkyl group, aC(═O)—NA₇-(C₁-C₆)alkyl group, a C(═O)—(C₁-C₆)alkyl-NA₇ group, aC(═O)—NA₇-(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, a C(═O)—NA₇-aryl group, aC(═O)—NA₇-heteroaryl group, a C(═O)—(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkylgroup, a C(═O)—(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, aC(═O)—(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, aC(═O)—(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alkyl group, aNA₈-(C₁-C₆)alkyl-NA₇ group, a NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkylgroup, a NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aNA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, aNA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aNA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, aNA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aNA₈-(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇ group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—NA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group or aC(═O)—NA₈-(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alkyl group;

-   -   A₇ representing a straight or branched, saturated or        unsaturated, optionally substituted C₁-C₁₆₀ hydrocarbon chain        wherein optionally at least one methylene unit is independently        replaced by —NHC(═O)—, —N(alkyl)C(═O)—, —C(═O)NH—,        —C(═O)N(alkyl)-, —OC(═O)—, —C(═O)O—, —OC(═O)O—, —CH(OH)—,        —CH(SO₃H)—, —O—, —C(═O)—, —S(═O)—, —S(═O)₂—, —NHS(═O)₂—,        —N(alkyl)S(═O)₂—, —S(═O)₂NH—, —S(═O)₂N(alkyl)-, —P(═O)(OH)—,        —P(═O)(OH)O—, —O—P(═O)(OH)—, —O—P(═O)(OH)—O— or a        heterocycloalkyl group optionally substituted with at least one        substituent, identical or different, chosen from —OH, —Oalkyl,        -alkyl, a halogen atom, —NH₂, —NHalkyl, and —N(alkyl)₂, being        understood that each A₇ comprising a SO₃H function can be under        salt forms such as alkali metal salts, for instance sodium salts        (SO₃ ⁻ ⁺Na);    -   A₈ representing a hydrogen atom or a (C₁-C₆)alkyl group, for        instance a hydrogen atom or a methyl group;    -   i representing an integer ranging from 1 to 50, for instance        ranging from 1 to 35, wherein said substituted amino acids        AA_(s) have the formula (VI):

wherein:

-   -   T represents a saturated or unsaturated, linear or branched,        (C₁—C) trivalent alkyl group, preferably T is

-   -   U group represents a single bond, —NHC(═O)—, —N(alkyl)C(═O)—,        —C(═O)NH—, —C(═O)N(alkyl)-, —NHC(═O)NH—, —NHC(═NH)NH—, —OC(═O)—,        —C(═O)O—, —OC(═O)O—, —S—, —Se—, —O—, —NH—, —N(alkyl)-, —C(═O)—,        —OP(═O)—, —S(═O)—, —S(═O)₂—, —NHS(═O)₂—, —N(alkyl)S(═O)₂—,        —S(═O)₂NH—, —S(═O)₂N(alkyl)-, —P(═O)(OH)—, —P(═O)(OH)O—,        —O—P(═O)(OH)—, or —O—P(═O)(OH)—O, such as U group represents        —NH—C(═O)—, or —C(═O)NH—; A₉ represents a straight or branched,        saturated or unsaturated, optionally substituted C₁-C₁₆₀        hydrocarbon chain wherein optionally at least one methylene unit        is independently replaced by —NH—C(═O)—, —N(alkyl)C(═O)—,        —C(═O)NH—, —C(═O)N(alkyl)-, —NHC(═NH)NH—, —OC(═O)—, —C(═O)O—,        —OC(═O)O—, —CH(OH)—, —CH(SO₃H)—, —CH(Oalkyl)-, —CHF—, —CF₂—,        —S—, —Se—, —O—, —NH—, —N(alkyl)-, —N⁺H(alkyl)-, —N⁺(alkyl)₂-,        —C(═O)—, —OP(═O)—, —S(═O)—, —S(═O)₂—, —NHS(═O)₂—,        —N(alkyl)S(═O)₂—, —S(═O)₂NH—, —S(═O)₂N(alkyl)-, —P(═O)(OH)—,        —P(═O)(OH)O—, —O—P(═O)(OH)—, —O—P(═O)(OH)—O— or a        heterocycloalkyl group optionally substituted with at least one        substituent, identical or different, chosen from —OH, —Oalkyl,        -alkyl, a halogen atom, —NH₂, —NHalkyl, and —N(alkyl)₂, for        instance A₉ is —[(CH₂)₂—O]₄—CH₃, —[(CH₂)₂—O]₂₄—CH₃,

or —[(CH₂)₂—O)]₄—(CH₂)₂—C(═O)—NH—(CH₂)₂—SO₃H;being understood that each A₉ comprising a SO₃H function can be undersalt forms such as alkali metal salts, for instance sodium salts (SO₃ ⁻⁺Na).

According to another particular embodiment, the disclosure relates tocompounds of formula (I):

RCG1-L-P  (I)

wherein

-   -   RCG1 represents a reactive chemical group that is reactive        towards a chemical group present on a polypeptide such as an        antibody;    -   P represents a hydrogen atom, —OH or an activated O;    -   L represents a linker of formula (II):

wherein:

-   -   L1 is of formula (III):

-   -   wherein:        -   when P represents a hydrogen atom, then x=0 or 1 and y=1 and            z=0;        -   when P represents —OH, then x=y=z=0;        -   when P represents an activated O, then x=1 and y=z=0, or            x=y=z=1;        -   J₁, J₂, J₃ and J₄ are chosen, independently of each other,            from CA₁ and N;        -   ALK represents a (C₁-C₁₂)alkylene group, for instance            (C₁-C₆)alkylene, such as of the form —(CH₂)_(n)—, n being an            integer ranging from 1 to 12 and for example ranging from 1            to 6;        -   A₁, A₂, A₃, A₄, A₅, and A₆ represent, independently of each            other, a hydrogen atom or a (C₁-C₆)alkyl group, such as a            hydrogen atom or a methyl group.    -   (AA)w represents a sequence of w substituted AA_(s) or        non-substituted amino acids AA_(ns) connected together via        peptide bonds;    -   w represents an integer ranging from 1 to 12, for instance from        1 to 6, such as 2 or 3;

if (AA)w contains at least one substituted amino acid AA_(s), then L2represents a single bond, a (C₁-C₆)alkyl group, a(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—O(C₁-C₆)alkyl group, a(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, a CH(SO₃H)—(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-CH(SO₃H) group, a (C₁-C₆)alkyl-cyclohexyl group, aC(═O)—(C₁-C₆)alkyl group, a C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—O(C₁-C₆)alkyl group, aC(═O)—(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, a C(═O)—CH(SO₃H)—(C₁-C₆)alkylgroup, a C(═O)—(C₁-C₆)alkyl-CH(SO₃H) group, aC(═O)—(C₁-C₆)alkyl-cyclohexyl group, a NA₈-(C₁-C₆)alkyl group, aNA₈-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aNA₈-(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—O(C₁-C₆)alkyl group, aNA₈-(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, a NA₈-(C₁-C₆)alkyl-CH(SO₃H) group,a C(═O)—NA₈-(C₁-C₆)alkyl group, a C(═O)—NA₈-(C₁-C₆)alkyl-(OCH₂CH₂)_(i)group, a C(═O)—NA₈-(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—O(C₁-C₆)alkyl group, aC(═O)—NA₈-(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-CH(SO₃H) group, a NA₇-(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-NA₇ group, a NA₇-(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, aNA₇-aryl group, a NA₇-heteroaryl group, a(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alkyl group, aC(═O)—NA₇-(C₁-C₆)alkyl group, a C(═O)—(C₁-C₆)alkyl-NA₇ group, aC(═O)—NA₇-(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, a C(═O)—NA₇-aryl group, aC(═O)—NA₇-heteroaryl group, a C(═O)—(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkylgroup, a C(═O)—(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, aC(═O)—(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, aC(═O)—(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alkyl group, aNA₈-(C₁-C₆)alkyl-NA₇ group, a NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkylgroup, a NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aNA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, aNA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aNA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, aNA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aNA₈-(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alky group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇ group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alky-(OCH₂CH₂)_(i) group, aC(═O)—NA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, aC(═O)—NA-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group or aC(═O)—NA₈-(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alkyl group;

if (AA)w represents a sequence of w non-substituted amino acids AA_(ns),then L2 represents a NA₇-(C₁-C₆)alkyl group, a (C₁-C₆)alkyl-NA₇ group, aNA₇-(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, a NA₇-aryl group, a NA₇-heteroarylgroup, a (C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alkyl group, aC(═O)—NA₇-(C₁-C₆)alkyl group, a C(═O)—(C₁-C₆)alkyl-NA₇ group, aC(═O)—NA₇-(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, a C(═O)—NA₇-aryl group, aC(═O)—NA₇-heteroaryl group, a C(═O)—(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkylgroup, a C(═O)—(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, aC(═O)—(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, aC(═O)—(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alkyl group, aNA₈-(C₁-C₆)alkyl-NA₇ group, a NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkylgroup, a NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aNA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, aNA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aNA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, aNA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aNA₈-(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alky group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇ group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—NA₆-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group or aC(═O)—NA₈-(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alkyl group;

-   -   A₇ representing a straight or branched, saturated or        unsaturated, optionally substituted C₁-C₁₆₀ hydrocarbon chain        wherein optionally at least one methylene unit is independently        replaced by —NHC(═O)—, —N(alkyl)C(═O)—, —C(═O)NH—,        —C(═O)N(alkyl)-, —OC(═O)—, —C(═O)O—, —OC(═O)O—, —CH(OH)—,        —CH(SO₃H)—, —O—, —C(═O)—, —S(═O)—, —S(═O)₂—, —NHS(═O)₂—,        —N(alkyl)S(═O)₂—, —S(═O)₂NH—, —S(═O)₂N(alkyl)-, —P(═O)(OH)—,        —P(═O)(OH)O—, —O—P(═O)(OH)—, —O—P(═O)(OH)—O— or a        heterocycloalkyl group optionally substituted with at least one        substituent, identical or different, chosen from —OH, —Oalkyl,        -alkyl, a halogen atom, —NH₂, —NHalkyl, and —N(alkyl)₂, being        understood that each A₇ comprising a SO₃H function can be under        salt forms such as alkali metal salts, for instance sodium salts        (SO₃ ⁻ ⁺Na);    -   A₈ representing a hydrogen atom or a (C₁-C₆)alkyl group, for        instance a hydrogen atom or a methyl group;    -   i representing an integer ranging from 1 to 50, for instance        ranging from 1 to 35, wherein said substituted amino acids        AA_(s) have the formula (VI):

wherein:

-   -   T represents a saturated or unsaturated, linear or branched,        (C₁-C₈) trivalent alkyl group, preferably T is

-   -   U group represents a single bond, —NHC(═O)—, —N(alkyl)C(═O)—,        —C(═O)NH—, —C(═O)N(alkyl)-, —NHC(═O)NH—, —NHC(═NH)NH—, —OC(═O)—,        —C(═O)O—, —OC(═O)O—, —S—, —Se—, —O—, —NH—, —N(alkyl)-, —C(═O)—,        —OP(═O)—, —S(═O)—, —S(═O)₂—, —NHS(═O)₂—, —N(alkyl)S(═O)₂—,        —S(═O)₂NH—, —S(═O)₂N(alkyl)-, —P(═O)(OH)—, —P(═O)(OH)O—,        —O—P(═O)(OH)—, or —O—P(═O)(OH)—O, such as U group represents        —NH—C(═O)—, or —C(═O)NH—;    -   A₉ represents a straight or branched, saturated or unsaturated,        optionally substituted C₁-C₁₆₀ hydrocarbon chain wherein        optionally at least one methylene unit is independently replaced        by —NH—C(═O)—, —N(alkyl)C(═O)—, —C(═O)NH—, —C(═O)N(alkyl)-,        —NHC(═NH)NH—, —OC(═O)—, —C(═O)O—, —OC(═O)O—, —CH(OH)—,        —CH(SO₃H)—, —CH(Oalkyl)-, —CHF—, —CF₂—, —S—, —Se—, —O—,        —N(alkyl)-, —N⁺H(alkyl)-, —N⁺(alkyl)₂-, —OP(═O)—, —S(═O)—,        —S(═O)₂—, —NHS(═O)₂—, —N(alkyl)S(═O)₂—, —S(═O)₂NH—,        —S(═O)₂N(alkyl)-, —P(═O)(OH)—, —P(═O)(OH)O—, —O—P(═O)(OH)—,        —O—P(═O)(OH)—O— or a heterocycloalkyl group optionally        substituted with at least one substituent, identical or        different, chosen from —OH, —Oalkyl, -alkyl, a halogen atom,        —NH₂, —NHalkyl, and —N(alkyl)₂, for instance A₉ is        —[(CH₂)₂—O]₄—CH₃, —[(CH₂)₂—O]₂₄—CH₃,

or —[(CH₂)₂—O)]₄—(CH₂)₂—C(═O)—NH—(CH₂)₂—SO₃H;being understood that each A₉ comprising a SO₃H function can be undersalt forms such as alkali metal salts, for instance sodium salts (SO₃ ⁻⁺Na).

According to another particular embodiment, the disclosure relates tocompounds of formula (I):

RCG1-L-P  (I)

wherein

-   -   RCG1 represents a reactive chemical group that is reactive        towards a chemical group present on a polypeptide such as an        antibody;    -   P represents a hydrogen atom, —OH or an activated O;    -   L represents a linker of formula (II):

wherein:

-   -   L1 is of formula (III):

-   -   wherein:        -   when P represents a hydrogen atom, then x=0 or 1 and y=1 and            z=0;        -   when P represents —OH, then x=y=z=0;        -   when P represents an activated O, then x=1 and y=z=0, or            x=y=z=1;        -   J₁, J₂, J₃ and J₄ are chosen, independently of each other,            from CA₁ and N;        -   ALK represents a (C₁-C₁₂)alkylene group, for instance            (C₁-C₆)alkylene, such as of the form —(CH₂)_(n)—, n being an            integer ranging from 1 to 12 and for example ranging from 1            to 6;        -   A₁, A₂, A₃, A₄, A₅, and A₆ represent, independently of each            other, a hydrogen atom or a (C₁-C₆)alkyl group, such as a            hydrogen atom or a methyl group.    -   (AA)w represents a sequence of w substituted AA_(s) or        non-substituted amino acids AA_(ns) connected together via        peptide bonds;    -   w represents an integer ranging from 1 to 12, for instance from        1 to 6, such as 2 or 3;

if (AA)w contains at least one substituted amino acid AA_(s), then L2represents a single bond, a (C₁-C₆)alkyl group, a(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—O(C₁-C₆)alkyl group, a(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, a CH(SO₃H)—(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-CH(SO₃H) group, a (C₁-C₆)alkyl-cyclohexyl group, aC(═O)—(C₁-C₆)alkyl group, a C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—O(C₁-C₆)alkyl group, aC(═O)—(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, a C(═O)—CH(SO₃H)—(C₁-C₆)alkylgroup, a C(═O)—(C₁-C₆)alkyl-CH(SO₃H) group, aC(═O)—(C₁-C₆)alkyl-cyclohexyl group, a NA₈-(C₁-C₆)alkyl group, aNA₈-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aNA₈-(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—O(C₁-C₆)alkyl group, aNA₈-(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, a NA₈-(C₁-C₆)alkyl-CH(SO₃H) group,a C(═O)—NA₈-(C₁-C₆)alkyl group, a C(═O)—NA₈-(C₁-C₆)alkyl-(OCH₂CH₂)_(i)group, a C(═O)—NA₈-(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—O(C₁-C₆)alkyl group, aC(═O)—NA₈-(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-CH(SO₃H) group, a NA₇-(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-NA₇ group, a NA₇-(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, aNA₇-aryl group, a NA₇-heteroaryl group, a(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alkyl group, aC(═O)—NA₇-(C₁-C₆)alkyl group, a C(═O)—(C₁-C₆)alkyl-NA₇ group, aC(═O)—NA₇-(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, a C(═O)—NA₇-aryl group, aC(═O)—NA₇-heteroaryl group, a C(═O)—(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkylgroup, a C(═O)—(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, aC(═O)—(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, aC(═O)—(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alkyl group, aNA₈-(C₁-C₆)alkyl-NA₇ group, a NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkylgroup, a NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aNA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, aNA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aNA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, aNA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aNA₈-(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alky group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇ group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alky-(OCH₂CH₂)_(i) group, aC(═O)—NA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group or aC(═O)—NA₈-(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alkyl group;

if (AA)w represents a sequence of w non-substituted amino acids AA_(ns),then L2 represents a NA₇-(C₁-C₆)alkyl group, a (C₁-C₆)alkyl-NA₇ group, aNA₇-(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, a NA₇-aryl group, a NA₇-heteroarylgroup, a (C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alkyl group, aC(═O)—NA₇-(C₁-C₆)alkyl group, a C(═O)—(C₁-C₆)alkyl-NA₇ group, aC(═O)—NA₇-(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, a C(═O)—NA₇-aryl group, aC(═O)—NA₇-heteroaryl group, a C(═O)—(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkylgroup, a C(═O)—(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, aC(═O)—(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, aC(═O)—(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alkyl group, aNA₈-(C₁-C₆)alkyl-NA₇ group, a NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkylgroup, a NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aNA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, aNA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aNA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, aNA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aNA₈-(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇ group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—NA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group or aC(═O)—NA₈-(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alkyl group;

-   -   A₇ representing a straight or branched, saturated or        unsaturated, optionally substituted C₁-C₁₆₀ hydrocarbon chain        wherein optionally at least one methylene unit is independently        replaced by —NHC(═O)—, —N(alkyl)C(═O)—, —C(═O)NH—,        —C(═O)N(alkyl)-, —OC(═O)—, —C(═O)O—, —OC(═O)O—, —CH(OH)—,        —CH(SO₃H)—, —O—, —C(═O)—, —S(═O)—, —S(═O)₂—, —NHS(═O)₂—,        —N(alkyl)S(═O)₂—, —S(═O)₂NH—, —S(═O)₂N(alkyl)-, —P(═O)(OH)—,        —P(═O)(OH)O—, —O—P(═O)(OH)—, —O—P(═O)(OH)—O— or a        heterocycloalkyl group optionally substituted with at least one        substituent, identical or different, chosen from —OH, —Oalkyl,        -alkyl, a halogen atom, —NH₂, —NHalkyl, and —N(alkyl)₂, being        understood that each A₇ comprising a SO₃H function can be under        salt forms such as alkali metal salts, for instance sodium salts        (SO₃ ⁻ ⁺Na);    -   A₈ representing a hydrogen atom or a (C₁-C₆)alkyl group, for        instance a hydrogen atom or a methyl group;    -   i representing an integer ranging from 1 to 50, for instance        ranging from 1 to 35, wherein said substituted amino acids        AA_(s) have the formula (VI):

wherein:

-   -   T represents a saturated or unsaturated, linear or branched,        (C₁-C₈) trivalent alkyl group, preferably T is

-   -   U group represents a single bond, —NHC(═O)—, —N(alkyl)C(═O)—,        —C(═O)NH—, —C(═O)N(alkyl)-, —NHC(═O)NH—, —NHC(═NH)NH—, —OC(═O)—,        —C(═O)O—, —OC(═O)O—, —S—, —Se—, —O—, —NH—, —N(alkyl)-, —C(═O)—,        —OP(═O)—, —S(═O)—, —S(═O)₂—, —NHS(═O)₂—, —N(alkyl)S(═O)₂—,        —S(═O)₂NH—, —S(═O)₂N(alkyl)-, —P(═O)(OH)—, —P(═O)(OH)O—,        —O—P(═O)(OH)—, or —O—P(═O)(OH)—O, such as U group represents        —NH—C(═O)—, or —C(═O)NH—;    -   A₉ represents a straight or branched, saturated or unsaturated,        optionally substituted C₁-C₁₆₀ hydrocarbon chain wherein        optionally at least one methylene unit is independently replaced        by —NH—C(═O)—, —N(alkyl)C(═O)—, —C(═O)NH—, —C(═O)N(alkyl)-,        —C(═O)O—, —OC(═O)O—, —CH(OH)—, —CH(SO₃H)—, —CH(Oalkyl)-, —CHF—,        —CF₂—, —Se—, —O—, —N(alkyl)-, —N⁺H(alkyl)-, —N⁺(alkyl)₂-,        —OP(═O)—, —S(═O)—, —S(═O)₂—, —NHS(═O)₂—, —N(alkyl)S(═O)₂—,        —S(═O)₂NH—, —S(═O)₂N(alkyl)-, —P(═O)(OH)—, —P(═O)(OH)O—,        —O—P(═O)(OH)—, —O—P(═O)(OH)—O— or a heterocycloalkyl group        optionally substituted with at least one substituent, identical        or different, chosen from —OH, —Oalkyl, -alkyl, a halogen atom,        —NH₂, —NHalkyl, and —N(alkyl)₂, for instance A₉ is        —[(CH₂)₂—O]₄—CH₃, —[(CH₂)₂—O]₂₄—CH₃,

or —[(CH₂)₂—O)]₄—(CH₂)₂—C(═O)—NH—(CH₂)₂—SO₃H;being understood that each A₉ comprising a SO₃H function can be undersalt forms such as alkali metal salts, for instance sodium salts (SO₃ ⁻⁺Na).

According to another particular embodiment, the disclosure relates tocompounds of formula (I):

RCG1-L-P  (I)

wherein

-   -   RCG1 represents a reactive chemical group that is reactive        towards a chemical group present on a polypeptide such as an        antibody;    -   P represents a hydrogen atom, —OH or an activated O;    -   L represents a linker of formula (II):

wherein:

-   -   L1 is of formula (III):

-   -   wherein:        -   when P represents a hydrogen atom, then x=0 or 1 and y=1 and            z=0;        -   when P represents —OH, then x=y=z=0;        -   when P represents an activated O, then x=1 and y=z=0, or            x=y=z=1;        -   J₁, J₂, J₃ and J₄ are chosen, independently of each other,            from CA₁ and N;        -   ALK represents a (C₁-C₁₂)alkylene group, for instance            (C₁-C₆)alkylene, such as of the form —(CH₂)_(n)—, n being an            integer ranging from 1 to 12 and for example ranging from 1            to 6;        -   A₁, A₂, A₃, A₄, A₅, and A₆ represent, independently of each            other, a hydrogen atom or a (C₁-C₆)alkyl group, such as a            hydrogen atom or a methyl group.    -   (AA)w represents a sequence of w substituted AA_(s) or        non-substituted amino acids AA_(ns) connected together via        peptide bonds;    -   w represents an integer ranging from 1 to 12, for instance from        1 to 6, such as 2 or 3;    -   (AA)w contains at least one substituted amino acid AA_(s) and/or        w non-substituted amino acid AA_(ns) and L2 represents a        (C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group;    -   A₇ is as defined above.

According to at least one embodiment, (AA)w contains at least onesubstituted amino acid AA_(s) and L2 represents:

-   -   a (C₁-C₆)alkyl group, such as a (CH₂)₃ group;    -   a C(═O)—(C₁-C₆)alkyl group, such as a C(═O)—(CH₂)₃ group; or    -   a (C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, such as a        (CH₂)₂—NA₇-(CH₂)₂ group in which A₇ is as defined above.

According to at least one embodiment, (AA)w contains at least onesubstituted amino acid AA_(s) and L2 represents:

-   -   a (C₁-C₆)alkyl group, such as a (CH₂)₃ group;    -   a C(═O)—(C₁-C₆)alkyl group, such as a C(═O)—(CH₂)₃ group; or    -   a (C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, such as a        (CH₂)₂—NA₇-(CH₂)₂ group in which A₇ is a        —C(═O)—(CH₂)₂—C(═O)—NH—(CH₂)₂—SO₃H group; being understood that        each A₇ comprising a SO₃H function can be under salt forms such        as alkali metal salts, for instance sodium salts (SO₃ ⁻ ⁺Na).

According to another embodiment, (AA)w contains w non-substituted aminoacid AA_(ns) and L2 represents:

a (C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, such as a (CH₂)₂—NA₇-(CH₂)₂ groupin which A₇ is as defined above.

According to another embodiment, (AA)w contains w non-substituted aminoacid AA_(ns) and L2 represents:

a (C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, such as a (CH₂)₂—NA₇-(CH₂)₂ groupin which A₇ represents:

-   -   a C(═O)—[(CH₂)₂—O]_(a)—CH₃ group wherein “a” represents an        integer ranging from 1 to 50, for instance ranging from 1 to 24,        such as 4, 7 and 24, for example A₇ is a C(═O)—[(CH₂)₂—O]₄—CH₃        group, a C(═O)—[(CH₂)₂—O]₇—CH₃ group, or a        C(═O)—[(CH₂)₂—O]₂₄—CH₃ group;    -   a —C(═O)—(CH₂)₂—C(═O)—NH—(CH₂)₂—SO₃H group; or    -   a        C(═O)—(CH₂)₂—C(═O)—NH—[(CH₂)₂—O]_(a)—(CH₂)₂—C(═O)—NH—(CH₂)₂—SO₃H        group wherein “a” represents an integer ranging from 1 to 50,        such as ranging from 1 to 24, for example 4, such as A₇ is        —C(═O)—(CH₂)₂—C(═O)—NH—[(CH₂)₂—O]₄—(CH₂)₂—C(═O)—NH—(CH₂)₂—SO₃H        group; being understood that each A₇ comprising a SO₃H function        can be under salt forms such as alkali metal salts, for instance        sodium salts (SO₃ ⁻ ⁺Na);

According to at least one embodiment, (AA)w contains at least onesubstituted amino acid AA_(s) and/or w non-substituted amino acidAA_(ns) and L2 represents:

a NA₇-(C₁-C₆)alkyl group, a (C₁-C₆)alkyl-NA₇ group, aNA₇-(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, a NA₇-aryl group, a NA₇-heteroarylgroup, a (C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alkyl group, aC(═O)—NA₇-(C₁-C₆)alkyl group, a C(═O)—(C₁-C₆)alkyl-NA₇ group, aC(═O)—NA₇-(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, a C(═O)—NA₇-aryl group, aC(═O)—NA₇-heteroaryl group, a C(═O)—(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkylgroup, a C(═O)—(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, aC(═O)—(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, aC(═O)—(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alkyl group, aNA₈-(C₁-C₆)alkyl-NA₇ group, a NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkylgroup, a NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aNA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, aNA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aNA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, aNA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aNA₈-(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇ group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—NA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group or aC(═O)—NA₆-(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alkyl group; wherein i,A₇ and A₈ are as defined above.

According to another embodiment, (AA)w contains at least one substitutedamino acid AA_(s) and/or w non-substituted amino acid AA_(ns) and L2represents:

a NA₇-(C₁-C₆)alkyl group, a (C₁-C₆)alkyl-NA₇ group, aNA₇-(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, a NA₇-aryl group, a NA₇-heteroarylgroup, a (C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alkyl group, aC(═O)—NA₇-(C₁-C₆)alkyl group, a C(═O)—(C₁-C₆)alkyl-NA₇ group, aC(═O)—NA₇-(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, a C(═O)—NA₇-aryl group, aC(═O)—NA₇-heteroaryl group, a C(═O)—(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkylgroup, a C(═O)—(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, aC(═O)—(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, aC(═O)—(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aNA₈-(C₁-C₆)alkyl-NA₇ group, a NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkylgroup, a NA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, aNA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, a C(═O)—NA₈-(C₁-C₆)alkyl-NA₇group, a C(═O)—NA-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, or aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group; wherein i, A₇ and A₈ areas defined above.

According to another embodiment, (AA)w contains at least one substitutedamino acid AA_(s) and/or w non-substituted amino acid AA_(ns) and L2represents:

a NA₇-(C₁-C₆)alkyl group, a (C₁-C₆)alkyl-NA₇ group, aNA₇-(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-(OCH₂CH₂)_(i)-NA₇-(C₁-C₆)alkyl group, aC(═O)—NA₇-(C₁-C₆)alkyl group, a C(═O)—(C₁-C₆)alkyl-NA₇ group, aC(═O)—NA₇-(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, aC(═O)—(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl group, aC(═O)—(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, aC(═O)—(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, aC(═O)—(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aNA₈-(C₁-C₆)alkyl-NA₇ group, a NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkylgroup, a NA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, aNA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, a C(═O)—NA₈-(C₁-C₆)alkyl-NA₇group, a C(═O)—NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, or aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group; wherein i, A₇ and A₈ areas defined above.

According to another embodiment, (AA)w contains at least one substitutedamino acid AA_(s) and/or w non-substituted amino acid AA_(ns) and L2represents:

a NA₇-(C₁-C₆)alkyl group, a (C₁-C₆)alkyl-NA₇ group, a(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, a C(═O)—NA₇-(C₁-C₆)alkyl group, aC(═O)—(C₁-C₆)alkyl-NA₇ group, a C(═O)—(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkylgroup, a C(═O)—(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, aC(═O)—(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, a NA₈-(C₁-C₆)alkyl-NA₇ group,a NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl group, aNA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, aNA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, a C(═O)—NA₈-(C₁-C₆)alkyl-NA₇group, a C(═O)—NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, or aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group; wherein A₇ and A₈ are asdefined above.

According to at least another embodiment, (AA)w contains at least onesubstituted amino acid AA_(s) and/or w non-substituted amino acidAA_(ns) and L2 comprises a A₇ representing:

a C(═O)—[(CH₂)₂—O]₄—CH₃ group; a C(═O)—[(CH₂)₂—O]₇—CH₃ group; aC(═O)—[(CH₂)₂—O]₂₄—CH₃ group; a C(═O)—(CH₂)₂—C(═O)—NH—(CH₂)₂—SO₃H group;a C(═O)—(CH₂)₂—C(═O)—NH—[(CH₂)₂—O]₄—(CH₂)₂—C(═O)—NH—(CH₂)₂—SO₃H group;being understood that each A₇ comprising a SO₃H function can be undersalt forms such as alkali metal salts, for instance sodium salts (SO₃ ⁻⁺Na).

According to at least another embodiment, (AA)w contains at least onesubstituted amino acid AA_(s) and/or w non-substituted amino acidAA_(ns) and L2 represents:

L1 may be one of the following (1111-5):

P Examples of L1 H -NA₆-ALK-NA₇- (III1)

(III2) OH — (III3) acti- vated O

(III4)

(III5)wherein:

-   -   J₁, J₂, J₃ and J₄ are chosen, independently of each other, from        CA₁ and N;    -   ALK represents a (C₁-C₁₂)alkylene group, for instance        (C₁-C₆)alkylene, such as of the form —(CH₂)_(n)—, n being an        integer ranging from 1 to 12 and for example ranging from 1 to        6;    -   A₁, A₂, A₃, A₄, A₅, and A₆ represent, independently of each        other, a hydrogen atom or a (C₁-C₆)alkyl group, such as a        hydrogen atom or a methyl group.

AA denotes an amino acid. An amino acid is a compound of formulaNH₂—CHA₁₀-COOH wherein A₁₀ represents the side chain of the AA. AA canbe a substituted AA_(s) or non-substituted AA_(ns) amino acid.

A non-substituted amino acid AA_(ns) denotes natural or non-naturalamino acid, of configuration D or L, identical to or derived from:alanine (Ala), β-alanine, γ-aminobutyric acid,2-amino-2-cyclohexylacetic acid, 2-amino-2-phenylacetic acid, arginine(Arg), asparagine (Asn), aspartic acid (Asp), citrulline (Cit), cysteine(Cys), α,α-dimethyl-γ-aminobutyric acid, β,β-dimethyl-γ-aminobutyricacid, glutamine (Gln), glutamic acid (Glu), glycine (Gly),homo-cysteine, selenocysteine, homo-selenocysteine, histidine (His),isoleucine (Ile), leucine (Leu), lysine (Lys), ε-acetyl-lysine (AcLys),methionine (Met), ornithine (Orn), phenylalanine (Phe), proline (Pro),serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), andvaline (Val).

For example, AA_(ns) represents alanine (Ala), citrulline (Cit), glycine(Gly), isoleucine (lie), leucine (Leu), lysine (Lys), ε-acetyl-lysine(AcLys), phenylalanine (Phe), tryptophan (Trp) and valine (Val).

The substituted amino acids AA_(s) have the formula (VI):

wherein:

-   -   T represents a saturated or unsaturated, linear or branched,        (C₁-C₈) trivalent alkyl group, for instance T is

-   -   U group represents a single bond, —NHC(═O)—, —N(alkyl)C(═O)—,        —C(═O)NH—, —C(═O)N(alkyl)-, —NHC(═O)NH—, —NHC(═NH)NH—, —OC(═O)—,        —C(═O)O—, —OC(═O)O—, —S—, —Se—, —O—, —NH—, —N(alkyl)-, —C(═O)—,        —OP(═O)—, —S(═O)—, —S(═O)₂—, —NHS(═O)₂—, —N(alkyl)S(═O)₂—,        —S(═O)₂NH—, —S(═O)₂N(alkyl)-, —P(═O)(OH)—, —P(═O)(OH)O—,        —O—P(═O)(OH)—, or —O—P(═O)(OH)—O, such as U group represents        —NH—C(═O)—, or —C(═O)NH—;    -   A₉ represents a straight or branched, saturated or unsaturated,        optionally substituted C₁-C₁₆₀ hydrocarbon chain wherein        optionally at least one methylene unit is independently replaced        by —NH—C(═O)—, —N(alkyl)C(═O)—, —C(═O)NH—, —C(═O)N(alkyl)-,        —NHC(═O)NH—, —NHC(═NH)NH—, —OC(═O)—, —C(═O)O—, —OC(═O)O—,        —CH(OH)—, —CH(SO₃H)—, —CH(Oalkyl)-, —CHF—, —CF₂—, —S—, —Se—,        —O—, —NH—, —N(alkyl)-, —N⁺H(alkyl)-, —N⁺(alkyl)₂-, —C(═O)—,        —OP(═O)—, —S(═O)—, —S(═O)₂—, —NHS(═O)₂—, —N(alkyl)S(═O)₂—,        —S(═O)₂NH—, —S(═O)₂N(alkyl)-, —P(═O)(OH)—, —P(═O)(OH)O—,        —O—P(═O)(OH)—, —O—P(═O)(OH)—O— or a heterocycloalkyl group        optionally substituted with at least one substituent, identical        or different, chosen from —OH, —Oalkyl, -alkyl, a halogen atom,        —NH₂, —NHalkyl, and —N(alkyl)₂, for instance A₉ is        [(CH₂)₂—O]₄—CH₃, [(CH₂)₂—O]₂₄—CH₃,

or [(CH₂)₂—O)]₄—(CH₂)₂—C(═O)—NH—(CH₂)₂—SO₃H;being understood that each A₉ comprising a SO₃H function can be undersalt forms such as alkali metal salts, for instance sodium salts (SO₃ ⁻⁺Na).

According to a particular embodiment, the substituted amino acids AA_(s)have the formula (VI):

wherein:

-   -   T represents a saturated or unsaturated, linear or branched,        (C₁-C₈) trivalent alkyl group, for instance T is

-   -   U group represents a single bond, —NHC(═O)—, —N(alkyl)C(═O)—,        —C(═O)NH—, —C(═O)N(alkyl)-, —NHC(═O)NH—, —NHC(═NH)NH—, —OC(═O)—,        —C(═O)O—, —OC(═O)O—, —S—, —Se—, —O—, —NH—, —N(alkyl)-, —C(═O)—,        —OP(═O)—, —S(═O)—, —S(═O)₂—, —NHS(═O)₂—, —N(alkyl)S(═O)₂—,        —S(═O)₂NH—, —S(═O)₂N(alkyl)-, —P(═O)(OH)—, —P(═O)(OH)O—,        —O—P(═O)(OH)—, or —O—P(═O)(OH)—O, such as U group represents        —NH—C(═O)—, or —C(═O)NH—;    -   A₉ represents a straight or branched, saturated or unsaturated,        optionally substituted C₁-C₁₆₀ hydrocarbon chain wherein        optionally at least one methylene unit is independently replaced        by —NH—C(═O)—, —N(alkyl)C(═O)—, —C(═O)NH—, —C(═O)N(alkyl)-,        —NHC(═NH)NH—, —OC(═O)—, —C(═O)O—, —OC(═O)O—, —CH(OH)—,        —CH(SO₃H)—, —CH(Oalkyl)-, —CHF—, —CF₂—, —S—, —Se—, —O—, —NH—,        —N(alkyl)-, —N⁺H(alkyl)-, —N⁺(alkyl)₂-, —C(═O)—, —OP(═O)—,        —S(═O)—, —S(═O)₂—, —NHS(═O)₂—, —N(alkyl)S(═O)₂—, —S(═O)₂NH—,        —S(═O)₂N(alkyl)-, —P(═O)(OH)—, —P(═O)(OH)O—, —O—P(═O)(OH)—,        —O—P(═O)(OH)—O— or a heterocycloalkyl group optionally        substituted with at least one substituent, identical or        different, chosen from —OH, —Oalkyl, -alkyl, a halogen atom,        —NH₂, —NHalkyl, and —N(alkyl)₂, for instance A₉ is        [(CH₂)₂—O]₄—CH₃, [(CH₂)₂—O]₂₄—CH₃,

or [(CH₂)₂—O)]₄—(CH₂)₂—C(═O)—NH—(CH₂)₂—SO₃H;being understood that each A₉ comprising a SO₃H function can be undersalt forms such as alkali metal salts, for instance sodium salts (SO₃ ⁻⁺Na).

According to another particular embodiment, the substituted amino acidsAA_(s) have the formula (VI):

wherein:

-   -   T represents a saturated or unsaturated, linear or branched,        (C₁-C₈) trivalent alkyl group, for instance T is

-   -   U group represents a single bond, —NHC(═O)—, —N(alkyl)C(═O)—,        —C(═O)NH—, —C(═O)N(alkyl)-, —NHC(═O)NH—, —NHC(═NH)NH—, —OC(═O)—,        —C(═O)O—, —OC(═O)O—, —S—, —Se—, —O—, —NH—, —N(alkyl)-, —C(═O)—,        —OP(═O)—, —S(═O)—, —S(═O)₂—, —NHS(═O)₂—, —N(alkyl)S(═O)₂—,        —S(═O)₂NH—, —S(═O)₂N(alkyl)-, —P(═O)(OH)—, —P(═O)(OH)O—,        —O—P(═O)(OH)—, or —O—P(═O)(OH)—O, such as U group represents        —NH—C(═O)—, or —C(═O)NH—;    -   A₉ represents a straight or branched, saturated or unsaturated,        optionally substituted C₁-C₁₆₀ hydrocarbon chain wherein        optionally at least one methylene unit is independently replaced        by —NH—C(═O)—, —N(alkyl)C(═O)—, —C(═O)NH—, —C(═O)N(alkyl)-,        —NHC(═NH)NH—, —OC(═O)—, —C(═O)O—, —OC(═O)O—, —CH(OH)—,        —CH(SO₃H)—, —CH(Oalkyl)-, —CHF—, —CF₂—, —S—, —Se—, —O—,        —N(alkyl)-, —N⁺H(alkyl)-, —N⁺(alkyl)₂-, —OP(═O)—, —S(═O)—,        —S(═O)₂—, —NHS(═O)₂—, —N(alkyl)S(═O)₂—, —S(═O)₂NH—,        —S(═O)₂N(alkyl)-, —P(═O)(OH)—, —P(═O)(OH)O—, —O—P(═O)(OH)—,        —O—P(═O)(OH)—O— or a heterocycloalkyl group optionally        substituted with at least one substituent, identical or        different, chosen from —OH, —Oalkyl, -alkyl, a halogen atom,        —NH₂, —NHalkyl, and —N(alkyl)₂, for instance A₉ is        [(CH₂)₂—O]₄—CH₃, [(CH₂)₂—O]₂₄—CH₃,

or [(CH₂)₂—O)]₄—(CH₂)₂—C(═O)—NH—(CH₂)₂—SO₃H;being understood that each A₉ comprising a SO₃H function can be undersalt forms such as alkali metal salts, for instance sodium salts (SO₃ ⁻⁺Na).

According to another particular embodiment, the substituted amino acidsAA_(s) have the formula (VI):

wherein:

-   -   T represents a saturated or unsaturated, linear or branched,        (C₁—C) trivalent alkyl group, for instance T is

-   -   U group represents a single bond, —NHC(═O)—, —N(alkyl)C(═O)—,        —C(═O)NH—, —C(═O)N(alkyl)-, —NHC(═O)NH—, —NHC(═NH)NH—, —OC(═O)—,        —C(═O)O—, —OC(═O)O—, —S—, —Se—, —O—, —NH—, —N(alkyl)-, —C(═O)—,        —OP(═O)—, —S(═O)—, —S(═O)₂—, —NHS(═O)₂—, —N(alkyl)S(═O)₂—,        —S(═O)₂NH—, —S(═O)₂N(alkyl)-, —P(═O)(OH)—, —P(═O)(OH)O—,        —O—P(═O)(OH)—, or —O—P(═O)(OH)—O, such as U group represents        —NH—C(═O)—, or —C(═O)NH—;    -   A₉ represents a straight or branched, saturated or unsaturated,        optionally substituted C₁-C₁₆₀ hydrocarbon chain wherein        optionally at least one methylene unit is independently replaced        by —NH—C(═O)—, —N(alkyl)C(═O)—, —C(═O)NH—, —C(═O)N(alkyl)-,        —C(═O)O—, —OC(═O)O—, —CH(OH)—, —CH(SO₃H)—, —CH(Oalkyl)-, —CHF—,        —CF₂—, —Se—, —O—, —N(alkyl)-, —N⁺H(alkyl)-, —N⁺(alkyl)₂-,        —OP(═O)—, —S(═O)—, —S(═O)₂—, —NHS(═O)₂—, —N(alkyl)S(═O)₂—,        —S(═O)₂NH—, —S(═O)₂N(alkyl)-, —P(═O)(OH)—, —P(═O)(OH)O—,        —O—P(═O)(OH)—, —O—P(═O)(OH)—O— or a heterocycloalkyl group        optionally substituted with at least one substituent, identical        or different, chosen from —OH, —Oalkyl, -alkyl, a halogen atom,        —NH₂, —NHalkyl, and —N(alkyl)₂, for instance A₉ is        [(CH₂)₂—O]₄—CH₃, [(CH₂)₂—O]₂₄—CH₃,

or [(CH₂)₂—O)]₄—(CH₂)₂—C(═O)—NH—(CH₂)₂—SO₃H;being understood that each A₉ comprising a SO₃H function can be undersalt forms such as alkali metal salts, for instance sodium salts (SO₃ ⁻⁺Na).

Examples of T that may be mentioned include:

According at least one embodiment, the substituted amino acids AA_(s)have the formula (VI) wherein:

-   -   T represents a saturated or unsaturated, linear or branched,        (C₁—C) trivalent alkyl group, such as

-   -   U group represents —NH—C(═O)—, or —C(═O)NH—;    -   A₉ represents a straight or branched, saturated or unsaturated,        optionally substituted C₁-C₁₆₀ hydrocarbon chain wherein        optionally at least one methylene unit is independently replaced        by —C(═O)NH—, —CH(OH)—, —CH(SO₃H)—, —O—, —NH—, or —C(═O)—, or a        heterocycloalkyl group optionally substituted with at least one        substituent, identical or different, chosen from —OH, —Oalkyl,        -alkyl, such as A₉ is [(CH₂)₂—O]₄—CH₃, [(CH₂)₂—O)]₂₄—CH₃,

or [(CH₂)₂—O]₄—(CH₂)₂—C(═O)—NH—(CH₂)₂—SO₃H;being understood that each A₉ comprising a SO₃H function can be undersalt forms such as alkali metal salts, for instance sodium salts (SO₃ ⁻⁺Na).

According to another embodiment, the substituted amino acids AA_(s) havethe formula (VI) wherein:

-   -   T represents

-   -   U group represents —NH—C(═O)—, or —C(═O)NH—;    -   A₉ represents [(CH₂)₂—O]₄—CH₃, [(CH₂)₂—O]₂₄—CH₃,

or [(CH₂)₂—O]₄—(CH₂)₂—C(═O)—NH—(CH₂)₂—SO₃H;being understood that each A₉ comprising a SO₃H function can be undersalt forms such as alkali metal salts, for instance sodium salts (SO₃ ⁻⁺Na).

According to a yet another embodiment, the substituted amino acidsAA_(s) have the formula (VI) wherein:

-   -   T represents

-   -   U group represents —NH—C(═O)—, or —C(═O)NH—;    -   A₉ represents —[(CH₂)₂—O]_(b)—CH₃ wherein “b” represents an        integer ranging from 1 to 50, for instance ranging from 1 to 24,        such as 4, 7 and 24, for example 4 or 24.

The sequence (AA)_(w) has the formula:

in which A₁₀ represents the side chain of one of the substituted AA_(s)or non-substituted AA_(ns) amino acids described above.

Examples of sequences of non-substituted amino acids (AA_(ns))w are asfollows: Gly-Gly, Phe-Lys, Val-Lys, Val-AcLys, Val-Cit, Phe-Phe-Lys,D-Phe-Phe-Lys, Gly-Phe-Lys, Ala-Lys, Val-Ala, Phe-Cit, Phe-Gly, Leu-Cit,Ile-Cit, Trp-Cit, Phe-Ala, Ala-Phe, Gly-Gly-Gly, Gly-Ala-Phe,Gly-Phe-Gly, Gly-Val-Cit, Gly-Phe-Leu-Cit (SEQ ID No. 3),Gly-Phe-Leu-Gly (SEQ ID No. 4), Ala-Leu-Ala-Leu (SEQ ID No. 5).

According to at least one embodiment, the sequence (AA_(ns))w ofnon-substituted amino acids AA_(ns) is selected from the following list:Gly-Gly, Phe-Lys, Val-Lys, Val-AcLys, Val-Cit, Phe-Phe-Lys,D-Phe-Phe-Lys, Gly-Phe-Lys, Ala-Lys, Val-Ala, Phe-Cit, Phe-Gly, Leu-Cit,Ile-Cit, Trp-Cit, Phe-Ala, Ala-Phe, Gly-Gly-Gly, Gly-Ala-Phe,Gly-Phe-Gly, Gly-Val-Cit, Gly-Phe-Leu-Cit (SEQ ID No. 3),Gly-Phe-Leu-Gly (SEQ ID No. 4), and Ala-Leu-Ala-Leu (SEQ ID No. 5), suchas Val-Ala and Val-Cit, for instance Val-Ala.

According to at least another embodiment, the sequence (AA_(s))wcontaining at least one substituted amino acid AA_(s) is selected fromthe list:

Among the linkers L of formula (II) that are the subject matter of thedisclosure, mention may be made for example of the following compounds:

Examples of RCG1 that may be mentioned include:

-   -   (i) a R_(a)Z_(a)—C(═O)— reactive group for which:    -   Z_(a) represents a single bond, —O— or —NH, such as —O—, and    -   R_(a) represents a hydrogen atom, a (C₁-C₆)alkyl, a        (C₃-C₇)cycloalkyl, an alkenyl, an aryl, a heteroaryl or a        (C₃-C₇)heterocycloalkyl group. The aryl group, the heteroaryl        group and/or the (C₃-C₇)heterocycloalkyl group may be        substituted by 1 to 5 atoms/groups chosen from a halogen atom,        such as a fluorine atom, an alkyl group, an alkoxy group, a        hydroxyl group, an oxo group, a nitro group and a cyano group;    -   (ii) one of the following reactive groups: a maleimido

group; a haloacetamido Cl, Br or

group with R₂₁ representing a hydrogen atom or a (C₁-C₆)alkyl group,such as Me; Cl—; N₃—; HO—; HS—; an activated disulfide such as H or

H₂N—; HC≡C— or an activated C≡C such as a cyclooctyne moiety forinstance a DBCO-amine

an O-alkyl hydroxylamine or a Pictet-Spengler reaction substrate such as

described in Agarwal P., et al., Bioconjugate Chem 2013, 24, 846-851.

According to a particular embodiment, RCG1 may be:

-   -   (i) a R_(a)Z_(a)—C(═O)— reactive group for which:    -   Z_(a) represents a single bond, —O— or —NH, such as —O—, and    -   R_(a) represents a hydrogen atom, a (C₁-C₆)alkyl, a        (C₃-C₇)cycloalkyl, an alkenyl, an aryl, a heteroaryl or a        (C₃-C₇)heterocycloalkyl group. The aryl group, the heteroaryl        group and/or the (C₃-C₇)heterocycloalkyl group may be        substituted by 1 to 5 atoms/groups chosen from a halogen atom,        such as a fluorine atom, an alkyl group, an alkoxy group, a        hydroxyl group, an oxo group, a nitro group and a cyano group;    -   (ii) one of the following reactive groups: a haloacetamido Cl,        Br or

group with R₂₁ representing a hydrogen atom or a (C₁-C₆)alkyl group,such as Me; Cl—; N₃—; HO—; HS—; an activated disulfide such as H or

H₂N—; HC≡C— or an activated C≡C such as a cyclooctyne moiety forinstance a DBCO-amine

an O-alkyl hydroxylamine or a Pictet-Spengler reaction substrate such as

described in Agarwal P., et al., Bioconjugate Chem 2013, 24, 846-851.

For instance, R_(a)Z_(a)— may represent HO—, CH₃O—, CH₂═CH—CH₂O—,

where cation represents for example sodium, potassium or cesium or,

group in which GI represents at least one electroinductive group such as—NO₂ or a halogen atom, such as a fluorine atom (F). They may be, forexample, the following groups:

Another type of R_(a)Z_(a)—C(═O)— group is the following:

For instance, RCG1 may be chosen from one of those described in theexamples that is to say chosen from the following groups:

COOH,

The present disclosure further relates to cryptophycin payloads offormula (IV):

wherein:

-   -   R₁ represents a (C₁-C₆)alkyl group,    -   R₂ and R₃ represent, independently of each other, a hydrogen        atom or a (C₁-C₆)alkyl group;    -    or alternatively R₂ and R₃ form together with the carbon atom        to which they are attached a (C₃-C₆)cycloalkyl or a        (C₃-C₆)heterocycloalkyl group;    -   R₄ and R₅ represent, independently of each other, a hydrogen        atom or a (C₁-C₆)alkyl group or a (C₁-C₆)alkyl-NH(R₁₂) group or        a (C₁-C₆)alkyl-OH group or a (C₁-C₆)alkyl-SH group or a        (C₁-C₆)alkyl-C(═O)₂H group;    -    or alternatively R₄ and R₅ form together with the carbon atom        to which they are attached a (C₃-C₆)cycloalkyl or a        (C₃-C₆)heterocycloalkyl group;    -   X represents O or N(R₆);    -   R₆ represents a hydrogen atom or a (C₁-C₆)alkyl group;    -   R₇ and R₈ represent, independently of each other, a hydrogen        atom or a (C₁-C₆)alkyl group or a (C₁-C₆)alkyl-C(═O)₂H group or        a (C₁-C₆)alkyl-N(C₁-C₆)alkyl₂ group;    -    or alternatively R₇ and R₈ form together with the carbon atom        to which they are attached a (C₃-C₆)cycloalkyl group or a        (C₃-C₆)heterocycloalkyl group;    -   R₉ represents at least one substituent of the phenyl nucleus        chosen, independently of each other, from: a hydrogen atom, —OH,        (C₁-C₄)alkoxy, a halogen atom, —NH₂, —NH(C₁-C₆)alkyl or        —N(C₁-C₆)alkyl₂ or —NH(C₁-C₆)cycloalkyl or        (C₃-C₆)heterocycloalkyl group;    -   R₁₀ represents at least one substituent of the phenyl nucleus        chosen from a hydrogen atom and a (C₁-C₄)alkyl group;    -   Y represents        -   —NR₁₁—(C₁-C₆)alkyl-, such as —NR₁₁—(CH₂)_(n)— like

-   -   -   —O—(C₁-C₆)alkyl-, such as —O—(CH₂)_(n)— like

-   -   -   —S—(C₁-C₆)alkyl-, such as —S—(CH₂)_(n)— like

Y being positioned in an ortho (o), meta (m) or para (p) position of thephenyl nucleus;

-   -   R₁₁ and R₁₂ represent, independently of each other, a hydrogen        atom or (C₁-C₆)alkyl, for instance a hydrogen atom or a methyl        group;    -   n represents an integer ranging from 1 to 6;    -   L is defined as in formula (I) and represents a linker of        formula (II) as defined in the present disclosure;    -   RCG1 represents a reactive chemical group present at the end of        the linker L, RCG1 being reactive towards a chemical group        present on a polypeptide such as an antibody. RCG1 is defined        above.

Each substituent R₁ to R₁₂ may also adopt one of the spatialconfigurations (e.g. R or S or alternatively Z or E) as described in theexamples. The compounds of formula (IV) may contain at least oneasymetric carbon atom. They may therefore exist in the form ofenantiomers or diastereoisomers. These enantiomers and diastereoisomers,and also mixtures thereof, including racemic mixtures, form part of thedisclosure.

As an example, the cryptophycin compound of formula (IV) may be one ofthe cryptophycin compounds of formula (I) described in WO2011/001052(such as one of D₁-D₈) or in PCT/EP2016/076603, (such as one of D₁-D19),as mentioned above.

Or the cryptophycin compound may be an equivalent unit described in oneof the examples.

Among the compounds of formula (IV) that are subject matter of thedisclosure, a group of compounds is composed of the compounds for whichR₁ represents a (C₁-C₆)alkyl, such as a methyl group.

Among the compounds of formula (IV) that are subject matter of thedisclosure, a group of compounds is composed of the compounds for whicheach of R₂ and R₃ represents a hydrogen atom.

Among the compounds of formula (IV) that are subject matter of thedisclosure, a group of compounds is composed of the compounds for whichone of R₂ and R₃ represents a (C₁-C₆)alkyl, such as a methyl group, andthe other one represents a hydrogen atom.

Among the compounds of formula (IV) that are subject matter of thedisclosure, a group of compounds is composed of the compounds for whichR₂ and R₃ form together with the carbon atom to which they are attacheda (C₃-C₆)cycloalkyl group, such as a cyclopropyl group.

Among the compounds of formula (IV) that are subject matter of thedisclosure, a group of compounds is composed of the compounds for whicheach of R₄ and R₅ represents a (C₁-C₆)alkyl, such as a methyl group.

Among the compounds of formula (IV) that are subject matter of thedisclosure, a group of compounds is composed of the compounds for whichX represents an oxygen atom, that is to say O.

Among the compounds of formula (IV) that are subject matter of thedisclosure, a group of compounds is composed of the compounds for whichX represents NH.

Among the compounds of formula (IV) that are subject matter of thedisclosure, a group of compounds is composed of the compounds for whichR₇ and R₈ represent independently of each other an hydrogen atom or a(C₁-C₆)alkyl group, such as an isobutyl group or a neopentyl group.

Among the compounds of formula (IV) that are subject matter of thedisclosure, a group of compounds is composed of the compounds for whichR₉ represents two substituents selected from a methoxy group and achlorine atom, such as the two R₉ substituents are 3-Cl and 4-methoxy.For instance, the phenyl nucleus comprises two substituents in positions3 and 4 on the phenyl nucleus.

Among the compounds of formula (IV) that are subject matter of thedisclosure, a group of compounds is composed of the compounds for whichR₁₀ represents a hydrogen atom.

Among the compounds of formula (IV) that are subject matter of thedisclosure, a group of compounds is composed of the compounds for whichY is positioned in the para position of the phenyl nucleus.

Among the compounds of formula (IV) that are subject matter of thedisclosure, a group of compounds is composed of the compounds for whichY represents NR₁₁—(C₁-C₆)alkyl, such as NR₁₁—(C₁-C₃)alkyl, for instanceNH—CH₂.

Among the compounds of formula (IV) that are subject matter of thedisclosure, a group of compounds is composed of the compounds for whichL of formula (II) comprises at least one substituted amino acid AA_(s)in the sequence of w amino acids (AA)w, L1 and L2 are as defined informula (II).

Among the compounds of formula (IV) that are subject matter of thedisclosure, a group of compounds is composed of the compounds for whichL of formula (II) comprises a sequence of w non-substituted amino-acidAA_(ns), L1 and L2 are as defined in formula (II).

Among the compounds of formula (IV) that are subject matter of thedisclosure, a group of compounds is composed of the compounds for which(AA)w in L of formula (II) contains at least one substituted amino acidAA_(s) and L2 represents:

-   -   a (C₁-C₆)alkyl group, such as a (CH₂)₃ group;    -   a C(═O)—(C₁-C₆)alkyl group, such as a C(═O)—(CH₂)₃ group; or    -   a (C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, such as a        (CH₂)₂—NA₇-(CH₂)₂ group in which A₇ is as defined above.

Among the compounds of formula (IV) that are subject matter of thedisclosure, a group of compounds is composed of the compounds for which(AA)w in L of formula (II) contains at least one substituted amino acidAA_(s) and L2 represents:

-   -   a (C₁-C₆)alkyl group, such as a (CH₂)₃ group;    -   a C(═O)—(C₁-C₆)alkyl group, such as a C(═O)—(CH₂)₃ group; or    -   a (C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, such as a        (CH₂)₂—NA₇-(CH₂)₂ group in which A₇ is a        —C(═O)—(CH₂)₂—C(═O)—NH—(CH₂)₂—SO₃H group;        being understood that each A₇ comprising a SO₃H function can be        under salt forms such as alkali metal salts, for instance sodium        salts (SO₃ ⁻ ⁺Na).

Among the compounds of formula (IV) that are subject matter of thedisclosure, a group of compounds is composed of the compounds for which(AA)w in L of formula (II) contains w non-substituted amino acid AA_(ns)and L2 represents:

a (C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, such as a (CH₂)₂—NA₇-(CH₂)₂ groupin which A₇ is as defined above.

Among the compounds of formula (IV) that are subject matter of thedisclosure, a group of compounds is composed of the compounds for which(AA)w in L of formula (II) contains w non-substituted amino acid AA_(ns)and L2 represents:

a (C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, such as a (CH₂)₂—NA₇-(CH₂)₂ groupin which A₇ represents:

-   -   a C(═O)—[(CH₂)₂—O]_(a)—CH₃ group wherein “a” represents an        integer ranging from 1 to 50, for instance ranging from 1 to 24,        such as 4, 7 and 24, for example A₇ is a C(═O)—[(CH₂)₂—O]₄—CH₃        group, a C(═O)—[(CH₂)₂—O]₇—CH₃ group, or a        C(═O)—[(CH₂)₂—O]₂₄—CH₃ group;    -   a —C(═O)—(CH₂)₂—C(═O)—NH—(CH₂)₂—SO₃H group; or    -   a        C(═O)—(CH₂)₂—C(═O)—NH—[(CH₂)₂—O]_(a)—(CH₂)₂—C(═O)—NH—(CH₂)₂—SO₃H        group wherein “a” represents an integer ranging from 1 to 50,        such as ranging from 1 to 24, for example 4, such as A₇ is        —C(═O)—(CH₂)₂—C(═O)—NH—[(CH₂)₂—O]₄—(CH₂)₂—C(═O)—NH—(CH₂)₂—SO₃H        group;        being understood that each A₇ comprising a SO₃H function can be        under salt forms such as alkali metal salts, for instance sodium        salts (SO₃ ⁻ ⁺Na);

Among the compounds of formula (IV) that are subject matter of thedisclosure, a group of compounds is composed of the compounds for which(AA)w in L of formula (II) contains at least one substituted amino acidAA_(s) and/or w non-substituted amino acid AA_(ns) and L2 represents:

a NA₇-(C₁-C₆)alkyl group, a (C₁-C₆)alkyl-NA₇ group, aNA₇-(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, a NA₇-aryl group, a NA₇-heteroarylgroup, a (C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alkyl group, aC(═O)—NA₇-(C₁-C₆)alkyl group, a C(═O)—(C₁-C₆)alkyl-NA₇ group, aC(═O)—NA₇-(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, a C(═O)—NA₇-aryl group, aC(═O)—NA₇-heteroaryl group, a C(═O)—(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkylgroup, a C(═O)—(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, aC(═O)—(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, aC(═O)—(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alkyl group, aNA₈-(C₁-C₆)alkyl-NA₇ group, a NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkylgroup, a NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aNA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, aNA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aNA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, aNA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aNA₈-(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇ group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—NA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group or aC(═O)—NA₈-(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alkyl group; wherein i,A₇ and A₈ are as defined above.

Among the compounds of formula (IV) that are subject matter of thedisclosure, a preferred group of compounds is composed of the compoundsfor which (AA)w in L of formula (II) contains at least one substitutedamino acid AA_(s) and/or w non-substituted amino acid AA_(ns) and L2represents:

a NA₇-(C₁-C₆)alkyl group, a (C₁-C₆)alkyl-NA₇ group, aNA₇-(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, a NA₇-aryl group, a NA₇-heteroarylgroup, a (C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alkyl group, aC(═O)—NA₇-(C₁-C₆)alkyl group, a C(═O)—(C₁-C₆)alkyl-NA₇ group, aC(═O)—NA₇-(CH₂CH₂O)(C₁-C₆)alkyl group, a C(═O)—NA₇-aryl group, aC(═O)—NA₇-heteroaryl group, a C(═O)—(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkylgroup, a C(═O)—(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, aC(═O)—(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, aC(═O)—(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aNA₈-(C₁-C₆)alkyl-NA₇ group, a NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkylgroup, a NA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, aNA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, a C(═O)—NA₈-(C₁-C₆)alkyl-NA₇group, a C(═O)—NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, or aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group; wherein i, A₇ and A₈ areas defined above.

Among the compounds of formula (IV) that are subject matter of thedisclosure, a more preferred group of compounds is composed of thecompounds for which (AA)w in L of formula (II) contains at least onesubstituted amino acid AA_(s) and/or w non-substituted amino acidAA_(ns) and L2 represents:

a NA₇-(C₁-C₆)alkyl group, a (C₁-C₆)alkyl-NA₇ group, aNA₇-(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-(OCH₂CH₂)_(i)-NA₇-(C₁-C₆)alkyl group, aC(═O)—NA₇-(C₁-C₆)alkyl group, a C(═O)—(C₁-C₆)alkyl-NA₇ group, aC(═O)—NA₇-(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, aC(═O)—(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl group, aC(═O)—(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, aC(═O)—(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, aC(═O)—(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aNA₈-(C₁-C₆)alkyl-NA₇ group, a NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkylgroup, a NA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, aNA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, a C(═O)—NA₈-(C₁-C₆)alkyl-NA₇group, a C(═O)—NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, or aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group; wherein i, A₇ and A₈ areas defined above.

Among the compounds of formula (IV) that are subject matter of thedisclosure, a still more preferred group of compounds is composed of thecompounds for which (AA)w in L of formula (II) contains at least onesubstituted amino acid AA_(s) and/or w non-substituted amino acidAA_(ns) and L2 represents:

a NA₇-(C₁-C₆)alkyl group, a (C₁-C₆)alkyl-NA₇ group, a(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, a C(═O)—NA₇-(C₁-C₆)alkyl group, aC(═O)—(C₁-C₆)alkyl-NA₇ group, a C(═O)—(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkylgroup, a C(═O)—(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, aC(═O)—(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, a NA₈-(C₁-C₆)alkyl-NA₇ group,a NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl group, aNA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, aNA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, a C(═O)—NA₈-(C₁-C₆)alkyl-NA₇group, a C(═O)—NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, or aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group; wherein A₇ and A₈ are asdefined above.

Among the compounds of formula (IV) that are subject matter of thedisclosure, a most preferred group of compounds is composed of thecompounds for which (AA)w in L of formula (II) contains at least onesubstituted amino acid AA_(s) and/or w non-substituted amino acidAA_(ns) and L2 represents:

a (C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group; wherein A₇ is as defined above.

Among the compounds of formula (IV) that are subject matter of thedisclosure, a group of compounds is composed of the compounds for which(AA)w in L of formula (II) contains at least one substituted amino acidAA_(s) and/or w non-substituted amino acid AA_(ns) and L2 comprises a A₇representing:

a C(═O)—[(CH₂)₂—O]₄—CH₃ group; a C(═O)—[(CH₂)₂—O]₇—CH₃ group; aC(═O)—[(CH₂)₂—O]₂₄—CH₃ group; a C(═O)—(CH₂)₂—C(═O)—NH—(CH₂)₂—SO₃H group;a C(═O)—(CH₂)₂—C(═O)—NH—[(CH₂)₂—O]₄—(CH₂)₂—C(═O)—NH—(CH₂)₂—SO₃H group;being understood that each A₇ comprising a SO₃H function can be undersalt forms such as alkali metal salts, for instance sodium salts (SO₃ ⁻⁺Na).

Among the compounds of formula (IV) that are subject matter of thedisclosure, a group of compounds is composed of the compounds for which(AA)w in L of formula (II) contains at least one substituted amino acidAA_(s) and/or w non-substituted amino acid AA_(ns) and L2 represents:

Among the compounds of formula (IV) that are subject matter of thedisclosure, a group of compounds is composed of the compounds comprisingin L a sequence (AA_(s))w containing at least one substituted amino acidAA_(s), (AA_(s))w being selected from the list:

Among the compounds of formula (IV) that are subject matter of thedisclosure, a group of compounds is composed of the compounds of thefollowing structure (beta epoxide configuration):

wherein:R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁, R₁₂, n, RCG₁, X, Y and Lare as defined in formula (IV).

All these sub-groups taken alone or in combination are part of thepresent disclosure.

According to at least one embodiment, the present disclosure relates tocompounds of formula (IV) wherein:

-   -   RCG1 is COOH,

-   -   L represents one of the below compounds of formula (II):

-   -   Y is a NR₁₁—(C₁₋₆)alkyl in which R₁₁ is a hydrogen atom, such as        Y is NH—CH₂;    -   R₁ represents a (C₁-C₆)alkyl group, such as a methyl group;    -   R₂ and R₃ represent independently of each other a hydrogen atom        or a (C₁-C₆)alkyl group such as a methyl group;    -   R₄ and R₅ represent independently of each other a (C₁-C₆)alkyl        group, such as a methyl group;    -   R₇ and R₈ represent independently of each other a hydrogen atom        or a (C₁-C₆)alkyl group, such as an isobutyl group or a        neopentyl group, for instance one of R₇ and R₈ represents a        (C₁-C₆)alkyl group, for example an isobutyl group or a neopentyl        group and the other of R₇ and R₈ represents a hydrogen atom;    -   X represents an oxygen atom or NH;    -   R₉ represents two substituents selected from a (C₁-C₄)alkoxy        group, such as a methoxy group, and a halogen atom, for instance        a chlorine atom, such as R₉ represents 3-Cl and 4-methoxy; and    -   R₁₀ represents a hydrogen atom.

Among the compounds of formula (IV) that are the subject matter of thepresent disclosure, mention may be made in particular of the followingcompounds:

The disclosure further relates to conjugates of formula (V):

wherein:

-   -   R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁ and R₁₂ are as        defined in formula (IV);    -   X, Y and L are as defined in formula (IV);    -   G represents the product of reaction between RCG1, a reactive        chemical group present at the end of the linker L of formula        (II), and RCG2, an orthogonal reactive chemical group present on        a polypeptide such as the antibody (Ab);    -   Ab represents an antibody.

The attachment between the cryptophycin payload of formula (IV) and theantibody, in order to obtain the conjugate of formula (V), is producedby the reaction between the reactive chemical group RCG1 present on thepayload as defined above that is reactive towards a reactive group RCG2present on the polypeptide such as the antibody. The reaction betweenRCG1 and RCG2 ensures the attachment of the compound of formula (IV) tothe antibody by formation of a covalent bond. In the conjugate offormula (V), parts of RCG1 and RCG2 can remain forming the attachmentbetween the linker and the antibody.

Examples of RCG2 that may be mentioned include (Garnett M. C., et al.,Advanced Drug Delivery Reviews 2001, 53, 171-216):

(i) ε-amino groups of lysines borne by the side chains of the lysineresidues that are present at the surface of an antibody;

(ii) α-amino groups of N-terminal amino acids of antibody heavy andlight chains;

(iii) the saccharide groups of the hinge region;

(iv) the thiols of cysteines generated by reducing intra-chain disulfidebonds or the thiols of engineered cysteines;

(v) amide groups borne by the side chains of some glutamine residuesthat are present at the surface of an antibody;

(vi) aldehyde groups introduced using formylglycine generating enzyme.

More recently, other conjugation approaches have been considered, forinstance the introduction of cysteines by mutation (Junutula J. R., etal., Nature Biotechnology 2008, 26, 925-932), the introduction ofunnatural amino acids allowing other types of chemistry (Axup J. Y., etal., PNAS 2012, 109, 40, 16101-16106) or the conjugation on antibodyglycans (Zhou Q., et al., Bioconjugate Chem. 2014, 25, 510-520). Use ofcysteine bridging dibromomaleimides (Behrens C. R., et al., Mol.Pharmaceutics 2015 3986-3998) and bis-sulfone reagents (Bryant P., etal., Mol. Pharmaceutics 2015 1872-1879) in order to cross-linkantibodies have also been described and could be applied to the presentdisclosure.

Another approach for site-specific modifications of antibodies is basedon enzymatic conjugation using for example bacterial transglutaminase(Jeger S., et al., Angew. Chem. Int. Ed. 2010, 49, 9995-9997; Strop P.,et al., Chem. Biol. 2013, 20, 161-167) or formylglycine generatingenzyme (Hudak J. E., et al., Angew. Chem. Int. Ed. 2012, 51, 4161-4165).For a review of site-specific conjugation strategies, see Agarwal P. andBertozzi C. R., Bioconjugate Chem 2015, 26, 176-192. These conjugationtechnologies may also be applied to cryptophycin payloads described inthe present disclosure.

It is also possible to chemically modify the polypeptide such as theantibody so as to introduce novel reactive chemical groups RCG2. Thus,it is well known to those skilled in the art how to modify an antibodywith the aid of a modifying agent introducing for example activateddisulfide, thiol, maleimido, haloacetamido, azido, alkyne or cycloalkynegroups (see especially WO2005/077090 page 14 and WO2011/001052). Themodification makes it possible to improve the conjugation reaction andto use a wider variety of RCG1 groups.

For instance, in the case where RCG1 is of the type (ii) above, it ispossible to chemically modify the antibody using an adequate modifyingagent or to introduce one or more unnatural amino acids so as tointroduce the adequate functions RCG2.

For example:

-   -   when RCG1 represents a N-hydroxysuccinimidyl ester, RCG2        represents a —NH₂ group;    -   when RCG1 represents a maleimido function, a haloacetamido        function, a chorine atom or an activated disulfide, RCG2 may be        a SH group;    -   when RCG1 represents a N₃ group, RCG2 may be a C≡CH function or        an activated C≡C function such as a cyclooctyne moiety;    -   when RCG1 represents a OH group or NH₂ group, RCG2 may be a        carboxylic acid or amide function;    -   when RCG1 represents a SH group, RCG2 may be a maleimido        function, a haloacetamido function or an activated disulfide        function;    -   when RCG1 represents a C≡CH function or an activated C≡C        function, RCG2 may be a N₃ group;    -   when RCG1 represents a O-alkyl hydroxylamine function or a        Pictet-Spengler reaction substrate, RCG2 may be an aldehyde or        ketone function.

Examples of G that may be mentioned include

For instance, G represents the following groups:

such as

Among the compounds of formula (V) that are subject matter of thepresent disclosure, a group of compounds is composed of the compoundsfor which R₁ represents a (C₁-C₆)alkyl, such as a methyl group.

Among the compounds of formula (V) that are subject matter of thedisclosure, a group of compounds is composed of the compounds for whicheach of R₂ and R₃ represents a hydrogen atom.

Among the compounds of formula (V) that are subject matter of thedisclosure, a group of compounds is composed of the compounds for whichone of R₂ and R₃ represents a (C₁-C₆)alkyl group, such as a methyl groupand the other one represents a hydrogen atom.

Among the compounds of formula (V) that are subject matter of thedisclosure, a group of compounds is composed of the compounds for whichR₂ and R₃ form together with the carbon atom to which they are attacheda (C₃-C₆)cycloalkyl group, such as a cyclopropyl group.

Among the compounds of formula (V) that are subject matter of thedisclosure, a group of compounds is composed of the compounds for whicheach of R₄ and R₅ represents a (C₁-C₆)alkyl group, in particular amethyl group.

Among the compounds of formula (V) that are subject matter of thedisclosure, a group of compounds is composed of the compounds for whichX represents an oxygen atom, that is to say O.

Among the compounds of formula (V) that are subject matter of thedisclosure, a group of compounds is composed of the compounds for whichX represents NH.

Among the compounds of formula (V) that are subject matter of thedisclosure, a group of compounds is composed of the compounds for whichR₇ and R₈ represent independently of each other a hydrogen atom or a(C₁-C₆)alkyl group, such as an isobutyl group or a neopentyl group.

Among the compounds of formula (V) that are subject matter of thedisclosure, a group of compounds is composed of the compounds for whichR₉ represents two substituents selected from a methoxy group and achlorine atom. For instance, the phenyl nucleus comprises twosubstituents in positions 3 and 4 on the phenyl nucleus. For example3-Cl and 4-methoxy.

Among the compounds of formula (V) that are subject matter of thedisclosure, a group of compounds is composed of the compounds for whichR₁₀ represents a hydrogen atom.

Among the compounds of formula (v) that are subject matter of thedisclosure, a group of compounds is composed of the compounds for whichY is positioned in the para position of the phenyl nucleus.

Among the compounds of formula (V) that are subject matter of thedisclosure, a group of compounds is composed of the compounds for whichY NR₁₁—(C₁-C₆)alkyl, such as NR₁₁—(C₁-C₃)alkyl, for instance NH—CH₂.

Among the compounds of formula (V) that are subject matter of thedisclosure, a group of compounds is composed of the compounds for whichL of formula (II) comprises at least one substituted amino acid AA_(s)in the sequence of w amino acids (AA)w, L1 and L2 are as defined informula (II).

Among the compounds of formula (V) that are subject matter of thedisclosure, a group of compounds is composed of the compounds for whichL of formula (II) comprises a sequence of w non-substituted amino-acidAA_(ns), L1 and L2 are as defined in formula (II).

Among the compounds of formula (V) that are subject matter of thedisclosure, a group of compounds is composed of the compounds for which(AA)w in L of formula (II) contains at least one substituted amino acidAA_(s) and L2 represents:

-   -   a (C₁-C₆)alkyl group, such as a (CH₂)₃ group;    -   a C(═O)—(C₁-C₆)alkyl group, such as a C(═O)—(CH₂)₃ group; or    -   a (C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, such as a        (CH₂)₂—NA₇-(CH₂)₂ group in which A₇ is as defined above.

Among the compounds of formula (V) that are subject matter of thedisclosure, a group of compounds is composed of the compounds for which(AA)w in L of formula (II) contains at least one substituted amino acidAA_(s) and L2 represents:

-   -   a (C₁-C₆)alkyl group, such as a (CH₂)₃ group;    -   a C(═O)—(C₁-C₆)alkyl group, such as a C(═O)—(CH₂)₃ group; or    -   a (C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, such as a        (CH₂)₂—NA₇-(CH₂)₂ group in which A₇ is a        —C(═O)—(CH₂)₂—C(═O)—NH—(CH₂)₂—SO₃H group;        being understood that each A₇ comprising a SO₃H function can be        under salt forms such as alkali metal salts, for instance sodium        salts (SO₃ ⁻ ⁺Na).

Among the compounds of formula (V) that are subject matter of thedisclosure, a group of compounds is composed of the compounds for which(AA)w in L of formula (II) contains w non-substituted amino acid AA_(ns)and L2 represents:

a (C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, such as a (CH₂)₂—NA₇-(CH₂)₂ groupin which A₇ is as defined above.

Among the compounds of formula (V) that are subject matter of thedisclosure, a group of compounds is composed of the compounds for which(AA)w in L of formula (II) contains w non-substituted amino acid AA_(ns)and L2 represents:

a (C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, such as a (CH₂)₂—NA₇-(CH₂)₂ groupin which A₇ represents:

-   -   a C(═O)—[(CH₂)₂—O]_(a)—CH₃ group wherein “a” represents an        integer ranging from 1 to 50, for instance ranging from 1 to 24,        such as 4, 7 and 24, for example A₇ is a C(═O)—[(CH₂)₂—O]₄—CH₃        group, a C(═O)—[(CH₂)₂—O]₇—CH₃ group, or a        C(═O)—[(CH₂)₂—O]₂₄—CH₃ group;    -   a —C(═O)—(CH₂)₂—C(═O)—NH—(CH₂)₂—SO₃H group; or    -   a        C(═O)—(CH₂)₂—C(═O)—NH—[(CH₂)₂—O]_(a)—(CH₂)₂—C(═O)—NH—(CH₂)₂—SO₃H        group wherein “a” represents an integer ranging from 1 to 50,        such as ranging from 1 to 24, for example 4, such as A₇ is        —C(═O)—(CH₂)₂—C(═O)—NH—[(CH₂)₂—O]₄—(CH₂)₂—C(═O)—NH—(CH₂)₂—SO₃H        group;        being understood that each A₇ comprising a SO₃H function can be        under salt forms such as alkali metal salts, for instance sodium        salts (SO₃ ⁻ ⁺Na); Among the compounds of formula (V) that are        subject matter of the disclosure, a group of compounds is        composed of the compounds for which (AA)w in L of formula (II)        contains at least one substituted amino acid AA_(s) and/or w        non-substituted amino acid AA_(ns) and L2 represents:        a NA₇-(C₁-C₆)alkyl group, a (C₁-C₆)alkyl-NA₇ group, a        NA₇-(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, a NA₇-aryl group, a        NA₇-heteroaryl group, a (C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl        group, a (C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group,        a (C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, a        (C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a        (C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, a        (C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a        (C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alkyl group, a        C(═O)—NA₇-(C₁-C₆)alkyl group, a C(═O)—(C₁-C₆)alkyl-NA₇ group, a        C(═O)—NA₇-(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, a C(═O)—NA₇-aryl        group, a C(═O)—NA₇-heteroaryl group, a        C(═O)—(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl group, a        C(═O)—(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a        C(═O)—(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, a        C(═O)—(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a        C(═O)—(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, a        C(═O)—(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a        C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alkyl group, a        NA₈-(C₁-C₆)alkyl-NA₇ group, a        NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl group, a        NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a        NA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, a        NA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a        NA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, a        NA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a        NA₈-(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alky group, a        C(═O)—NA₈-(C₁-C₆)alkyl-NA₇ group, a        C(═O)—NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl group, a        C(═O)—NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i)        group, a C(═O)—NA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, a        C(═O)—NA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i)        group, a C(═O)—NA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, a        C(═O)—NA-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group or a        C(═O)—NA₈-(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alkyl group;        wherein i, A₇ and A₈ are as defined above.

Among the compounds of formula (V) that are subject matter of thedisclosure, a preferred group of compounds is composed of the compoundsfor which (AA)w in L of formula (II) contains at least one substitutedamino acid AA_(s) and/or w non-substituted amino acid AA_(ns) and L2represents:

a NA₇-(C₁-C₆)alkyl group, a (C₁-C₆)alkyl-NA₇ group, aNA₇-(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, a NA₇-aryl group, a NA₇-heteroarylgroup, a (C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alkyl group, aC(═O)—NA₇-(C₁-C₆)alkyl group, a C(═O)—(C₁-C₆)alkyl-NA₇ group, aC(═O)—NA₇-(CH₂CH₂O)(C₁-C₆)alkyl group, a C(═O)—NA₇-aryl group, aC(═O)—NA₇-heteroaryl group, a C(═O)—(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkylgroup, a C(═O)—(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, aC(═O)—(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, aC(═O)—(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aNA₈-(C₁-C₆)alkyl-NA₇ group, a NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkylgroup, a NA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, aNA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, a C(═O)—NA₈-(C₁-C₆)alkyl-NA₇group, a C(═O)—NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, or aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group; wherein i, A₇ and A₈ areas defined above.

Among the compounds of formula (V) that are subject matter of thedisclosure, a more preferred group of compounds is composed of thecompounds for which (AA)w in L of formula (II) contains at least onesubstituted amino acid AA_(s) and/or w non-substituted amino acidAA_(ns) and L2 represents:

a NA₇-(C₁-C₆)alkyl group, a (C₁-C₆)alkyl-NA₇ group, aNA₇-(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-(OCH₂CH₂)_(i)-NA₇-(C₁-C₆)alkyl group, aC(═O)—NA₇-(C₁-C₆)alkyl group, a C(═O)—(C₁-C₆)alkyl-NA₇ group, aC(═O)—NA₇-(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, aC(═O)—(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl group, aC(═O)—(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, aC(═O)—(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, aC(═O)—(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aNA₈-(C₁-C₆)alkyl-NA₇ group, a NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkylgroup, a NA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, aNA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, a C(═O)—NA₈-(C₁-C₆)alkyl-NA₇group, a C(═O)—NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, or aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group; wherein i, A₇ and A₈ areas defined above.

Among the compounds of formula (V) that are subject matter of thedisclosure, a still more preferred group of compounds is composed of thecompounds for which (AA)w in L of formula (II) contains at least onesubstituted amino acid AA_(s) and/or w non-substituted amino acidAA_(ns) and L2 represents:

a NA₇-(C₁-C₆)alkyl group, a (C₁-C₆)alkyl-NA₇ group, a(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, a C(═O)—NA₇-(C₁-C₆)alkyl group, aC(═O)—(C₁-C₆)alkyl-NA₇ group, a C(═O)—(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkylgroup, a C(═O)—(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, aC(═O)—(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, a NA₈-(C₁-C₆)alkyl-NA₇ group,a NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl group, aNA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, aNA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, a C(═O)—NA₈-(C₁-C₆)alkyl-NA₇group, a C(═O)—NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, or aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group; wherein A₇ and A₈ are asdefined above.

Among the compounds of formula (V) that are subject matter of thedisclosure, a most preferred group of compounds is composed of thecompounds for which (AA)w in L of formula (II) contains at least onesubstituted amino acid AA_(s) and/or w non-substituted amino acidAA_(ns) and L2 represents:

a (C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group; wherein A₇ is as defined above.

Among the compounds of formula (V) that are subject matter of thedisclosure, a group of compounds is composed of the compounds for which(AA)w in L of formula (II) contains at least one substituted amino acidAA_(s) and/or w non-substituted amino acid AA_(ns) and L2 comprises a A₇representing:

a C(═O)—[(CH₂)₂—O]₄—CH₃ group; a C(═O)—[(CH₂)₂—O]₇—CH₃ group; aC(═O)—[(CH₂)₂—O]₂₄—CH₃ group; a C(═O)—(CH₂)₂—C(═O)—NH—(CH₂)₂—SO₃H group;a C(═O)—(CH₂)₂—C(═O)—NH—[(CH₂)₂—O]₄—(CH₂)₂—C(═O)—NH—(CH₂)₂—SO₃H group;being understood that each A₇ comprising a SO₃H function can be undersalt forms such as alkali metal salts, for instance sodium salts (SO₃ ⁻⁺Na).

Among the compounds of formula (V) that are subject matter of thedisclosure, a group of compounds is composed of the compounds for which(AA)w in L of formula (II) contains at least one substituted amino acidAA_(s) and/or w non-substituted amino acid AA_(ns) and L2 represents:

Among the compounds of formula (V) that are subject matter of thedisclosure, a group of compounds is composed of the compounds comprisingin L a sequence (AA_(s))w containing at least one substituted amino acidAA_(s), (AA_(s))w being selected from the list:

Among the compounds of formula (V) that are subject matter of thedisclosure, a group of compounds is composed of the compounds for whichAb is an anti-Epha2 antibody, for instance hu2H11_R35R74 antibody asrepresented by SEQ ID NO: 1 (light chain of antibody hu2H11_R35R74) andby SEQ ID NO: 2 (heavy chain of antibody hu2H11_R35R74) corresponding torespectively SEQ ID NO: 16 and SEQ ID NO:18 represented in WO2011039724A1.

According to at least one embodiment, the present disclosure relates tocompounds of formula (V) wherein:

-   -   Ab represents an antibody;    -   G represents:

-   -   Y is a NR₁₁—(C₁-6)alkyl group in which R₁₁ is a hydrogen atom,        for instance Y is a NH—CH₂ group;    -   L represents:

-   -   R₁ represents a (C₁-C₆)alkyl group, such as a methyl group;    -   R₂ and R₃ represent independently of each other a hydrogen atom        or a (C₁-C₆)alkyl group such as a methyl group;    -   R₄ and R₅ represent independently of each other a (C₁-C₆)alkyl        group, such as a methyl group;    -   R₇ and R₈ represent independently of each other a hydrogen atom        or a (C₁-C₆)alkyl group, such as an isobutyl group or a        neopentyl group, for instance one of R₇ and R₈ represents a        (C₁-C₆)alkyl group, such as an isobutyl group or a neopentyl        group and the other of R₇ and R₈ represents a hydrogen atom;    -   X represents an oxygen atom or NH;    -   R₉ represents two substituents selected from a (C₁-C₄)alkoxy        group, such as a methoxy group, and a halogen atom, such as a        chlorine atom, for instance R₉ represents 3-Cl and 4-methoxy;        and    -   R₁₀ represents a hydrogen atom.

Among the compounds of formula (V) that are subject matter of thedisclosure, a group of compounds is composed of the compounds of thefollowing structure (beta epoxide configuration):

wherein:

R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁, R₁₂, n, RCG₁, X, Y and Lare as defined in formula (IV).

All these sub-groups taken alone or in combination are part of thepresent disclosure.

Among the compounds of formula (V) that are the subject matter of thedisclosure, mention may be made of the following compounds:

wherein Ab is as defined in formula (V) of the present disclosure.

In accordance with the disclosure, the compounds of general formula (I),(11), (Ill), (IV) and (V) can be prepared by the following processes.

Preparation of the Linkers of Formula (II)

Preparation of the Spacers with Improved Hydrophilicity

Step (i): protection of the carboxylic acid as an allyl ester usingallyl bromide and a base such as, for example, cesium carbonate;

Step (ii): deprotection of the Boc amine using a solution of HCl (forexample solution in dioxane) or of TFA;

Step (iii): addition of the amine on tert-butyl acrylate in the presenceof a base such as, for example, DIEA;

Step (iv): coupling between the amine and an activated carboxylic acidsuch as a NHS ester or an acyl chloride in the presence of a base suchas, for example, DIEA;

Step (v): deprotection of the tert-butyl ester using a solution of HCl(for example solution in dioxane) or of TFA;

Step (vi): activation of the carboxylic acid as a NHS ester by treatmentwith DSC in the presence of a base such as, for example, DIEA;

Step (vii): coupling to succinic anhydride;

Step (viii): activation of the carboxylic acid as a NHS ester bytreatment with DSC in the presence of a base such as, for example, DIEAand coupling with an amine.

Scheme 1 depicted the synthesis starting with Boc-R₃-Ala-OH (CAS number[3303-84-2]) but may also apply to other Boc protected amino-alkyl acidswhich are commercially available for n ranging from 3 to 10. It depictedthe synthesis using succinic anhydride but may also apply to glutaricanhydre or alkyl diacids which are commercially available for n rangingfrom 3 to 10.

Combination of the Spacers with Improved Hydrophilicity and theClassical Dipeptides

Step (i): peptidic coupling using coupling reagents such as, forexample, EDC and HOBt and deprotection of the Fmoc amine in the presenceof a base such as, for example, piperidine;

Step (ii): for spacer 3, activation of the carboxylic acid as a NHSester by treatment with DSC in the presence of a base such as, forexample, DIEA and coupling of spacers 3 and 4 with the dipeptide;

Step (iii): deprotection of the tert-butyl ester using a solution of HCl(for example solution in dioxane) or of TFA;

Step (iv): activation of the carboxylic acid as a NHS ester by treatmentwith DSC in the presence of a base such as, for example, DIEA andcoupling with the amine;

Step (v): deprotection of the Boc amine using a solution of HCl (forexample solution in dioxane) or of TFA.

Scheme 2 depicted the synthesis of linkers using a Val-Ala dipeptide butmay also apply to other dipeptides; it depicted the synthesis usingspacers 3 or 4 but may also apply to spacers 1 or 2; it depicted thesynthesis using Boc-monoprotected ethylenediamine but may also apply toother Boc-monoprotected diamines which are commercially available for nranging from 3 to 10.

Preparation of the Dipeptides with Improved Hydrophilicity

Preparation of the Dipeptides with Improved Hydrophilicity Based onAmino Acids Bearing a Carboxylic Acid Group on their Side Chain

Step (i): peptidic coupling between the carboxylic acid and an amineusing coupling reagents such as, for example, EDC and HOBt;

Step (ii): activation of the carboxylic acid as a NHS ester by treatmentwith DSC in the presence of a base such as, for example, DIEA;

Step (iii): peptidic coupling between the NHS ester and an amine in thepresence of a base such as, for example, sodium bicarbonate or DIEA;

Step (iv): deprotection of the tert-butyl ester using a solution of HCl(for example solution in dioxane) or of TFA;

Step (v): peptidic coupling of the dipeptide with Boc-monoprotectedethylene diamine using a coupling reagent such as for example, T3P;

Step (vi): deprotection of the Boc amine using a solution of HCl (forexample solution in dioxane) or of TFA;

Step (vii): peptidic coupling of the dipeptide with p-aminobenzylalcohol using coupling reagents such as, for example, EEDQ;

Step (viii): activation of the benzylic alcohol as a p-nitrophenylcarbonate by treatment with p-nitrophenyl-chloroformate in the presenceof a base such as, for example, DIEA;

Step (ix): formation of a carbamate between the activated alcohol andthe amine in the presence of a base such as, for example, DIEA.

Scheme 3 depicted the synthesis of dipeptides starting with Val but mayapply to other amino acids listed above; it depicted the synthesis usingL-Glu tert-butyl ester (CAS number [45120-30-7]) but may also apply toother amino acids bearing a carboxylic acid on their side chain such as,for example, L-Asp tert-butyl ester (CAS number [4125-93-3]), D-Asptert-butyl ester (CAS number [148823-36-3]), D-Glu tert-butyl ester (CASnumber [25456-76-2]), 2-amino-hexanedioic acid 1-tert-butyl ester (CASnumber [1245806-58-9]) or 2-amino-heptanedioic acid 1-tert-butyl ester(CAS number [1888498-03-0]). Scheme 4 depicted the synthesis ofdipeptides using p-aminobenzyl alcohol (CAS number [623-04-1]) but mayalso apply to other aminobenzyl alcohol compounds which are commerciallyavailable such as, for example, 4-(1-hydroxyethyl)-aniline (racemic (CASnumber [14572-89-5]) or enantiopure (R) (CAS number [210754-25-9]) or(S) (CAS number [500229-84-5])), 4-amino-α,α-dimethyl-benzene-methanol(CAS number [23243-04-1]), 4-amino-α-methoxy-α-methyl-benzenemethanol(CAS number [1379318-81-6]),4-amino-α-methyl-α-trifluoromethyl-benzenemethanol (CAS number[851652-56-7]), 2-amino-benzenemethanol (CAS number [5344-90-1]),2-amino-α-methyl-benzenemethanol (racemic (CAS number [10517-50-7]) orenantiopure (R) (CAS number [3205-21-8]) or (S) (CAS number[3205-21-8])), 6-amino-3-pyridinemethanol (CAS number [113293-71-3]),6-amino-α-methyl-3-pyridinemethanol (CAS number [1335054-83-5]),6-amino-α-ethyl-3-pyridinemethanol (CAS number [1355225-85-2]),6-amino-α,α-dimethyl-3-pyridinemethanol (CAS number [843646-03-8]),5-amino-3-pyridinemethanol (CAS number [873651-92-4]),2-amino-3-pyridinemethanol (CAS number [23612-57-9]),2-amino-α-methyl-3-pyridinemethanol (racemic (CAS number [869567-91-9])or enantiopure (R) (CAS number [936718-01-3]) or (S) (CAS number[936718-00-2])), 2-amino-α-ethyl-3-pyridinemethanol (CAS number[914223-90-8]), 2-amino-α,α-dimethyl-3-pyridinemethanol (CAS number[213666-96-7]), 3-amino-4-pyridinemethanol (CAS number [152398-05-5]),3-amino-α-methyl-4-pyridinemethanol (CAS number [1242470-88-7]),3-amino-α,α-methyl-4-pyridinemethanol (CAS number [13357-81-8]),4-amino-3-pyridinemethanol (CAS number [138116-34-4]),4-amino-α-methyl-3-pyridinemethanol (CAS number [741223-49-4]),4-amino-α,α-methyl-3-pyridinemethanol (CAS number [1339013-26-1]),3-amino-2-pyridinemethanol (CAS number [52378-63-9]),3-amino-α-methyl-2-pyridinemethanol (CAS number [954240-54-1]),3-amino-α,α-methyl-2-pyridinemethanol (CAS number [899438-57-4]); itdepicted the synthesis using Boc-monoprotected ethylenediamine but mayalso apply to other Boc-monoprotected diamines which are commerciallyavailable for n ranging from 3 to 10.

Preparation of the Dipeptides with Improved Hydrophilicity Based onAmino Acids Bearing an Amino Group on their Side Chain

Or alternatively dipeptide 5 can be prepared following the synthesisdescribed in Scheme 6.

Step (i): peptidic coupling between the carboxylic acid and an amineusing coupling reagents such as, for example, EDC and HOBt;

Step (ii): activation of the carboxylic acid as a NHS ester by treatmentwith DSC in the presence of a base such as, for example, DIEA;

Step (iii): peptidic coupling between the NHS ester and an amine in thepresence of a base such as, for example, sodium bicarbonate or DIEA;

Step (iv): deprotection of the Boc amine using a solution of HCl (forexample solution in dioxane) or of TFA;

Step (v): peptidic coupling of the dipeptide with Boc-monoprotectedethylene diamine using a coupling reagent such as for example, T3P;

Step (vi): peptidic coupling of the dipeptide with p-aminobenzyl alcoholusing coupling reagents such as, for example, EEDQ;

Step (vii): activation of the benzylic alcohol as a p-nitrophenylcarbonate by treatment with p-nitrophenyl-chloroformate in the presenceof a base such as, for example, DIEA;

Step (viii): formation of a carbamate between the activated alcohol andthe amine in the presence of a base such as, for example, DIEA.

Schemes 5 and 6 depicted the synthesis of dipeptides starting with Valbut may apply to other amino acids listed above. Scheme 5 depicted thesynthesis using L-Lys Boc-protected on the side chain (CAS number[2418-95-3]) but may also apply to other amino acids bearing an aminogroup on their side chain such as, for example,(S)-3-amino-2-(tert-butoxycarbonylamino)propanoic acid (CAS number[73259-81-1]), (S)-3-amino-2-(tert-butoxycarbonylamino)propanoic acid(CAS number [76387-70-7]),(S)-4-amino-2-(tert-butoxycarbonylamino)butanoic acid (CAS number[25691-37-6]), (R)-4-amino-2-(tert-butoxycarbonylamino)butanoic acid(CAS number [80445-78-9]), L-ornithine Boc-protected on the side chain(CAS number [21887-64-9]), D-ornithine Boc-protected on the side chain(CAS number [159877-12-0]), D-Lys Boc-protected on the side chain (CASnumber [106719-44-2]),(2S)-2-amino-7-[[(1,1-dimethylethoxy)carbonyl]amino]heptanoic acid (CASnumber [1142814-17-2]) or(2R)-2-amino-7-[[(1,1-dimethylethoxy)carbonyl]amino]heptanoic acid (CASnumber [117833-90-6]). Scheme 7 depicted the synthesis of dipeptidesusing p-aminobenzyl alcohol (CAS number [623-04-1]) but may also applyto other aminobenzyl alcohol compounds which are commercially availablesuch as, for example, 4-(1-hydroxyethyl)-aniline (racemic (CAS number[14572-89-5]) or enantiopure (R) (CAS number [210754-25-9]) or (S) (CASnumber [500229-84-5])), 4-amino-α,α-dimethyl-benzene-methanol (CASnumber [23243-04-1]), 4-amino-α-methoxy-α-methyl-benzenemethanol (CASnumber [1379318-81-6]),4-amino-α-methyl-α-trifluoromethyl-benzenemethanol (CAS number[851652-56-7]), 2-amino-benzenemethanol (CAS number [5344-90-1]),2-amino-α-methyl-benzenemethanol (racemic (CAS number [10517-50-7]) orenantiopure (R) (CAS number [3205-21-8]) or (S) (CAS number[3205-21-8])), 6-amino-3-pyridinemethanol (CAS number [113293-71-3]),6-amino-α-methyl-3-pyridinemethanol (CAS number [1335054-83-5]),6-amino-α-ethyl-3-pyridinemethanol (CAS number [1355225-85-2]),6-amino-α,α-dimethyl-3-pyridinemethanol (CAS number [843646-03-8]),5-amino-3-pyridinemethanol (CAS number [873651-92-4]),2-amino-3-pyridinemethanol (CAS number [23612-57-9]),2-amino-α-methyl-3-pyridinemethanol (racemic (CAS number [869567-91-9])or enantiopure (R) (CAS number [936718-01-3]) or (S) (CAS number[936718-00-2])), 2-amino-α-ethyl-3-pyridinemethanol (CAS number[914223-90-8]), 2-amino-α,α-dimethyl-3-pyridinemethanol (CAS number[213666-96-7]), 3-amino-4-pyridinemethanol (CAS number [152398-05-5]),3-amino-α-methyl-4-pyridinemethanol (CAS number [1242470-88-7]),3-amino-α,α-methyl-4-pyridinemethanol (CAS number [13357-81-8]),4-amino-3-pyridinemethanol (CAS number [138116-34-4]),4-amino-α-methyl-3-pyridinemethanol (CAS number [741223-49-4]),4-amino-α,α-methyl-3-pyridinemethanol (CAS number [1339013-26-1]),3-amino-2-pyridinemethanol (CAS number [52378-63-9]),3-amino-α-methyl-2-pyridinemethanol (CAS number [954240-54-1]),3-amino-α,α-methyl-2-pyridinemethanol (CAS number [899438-57-4]); itdepicted the synthesis using Boc-monoprotected ethylenediamine but mayalso apply to other Boc-monoprotected diamines which are commerciallyavailable for n ranging from 3 to 10.

Preparation of the Cryptophycin Compounds

Cryptophycin compounds may be prepared as described in WO2011/001052 forX═O and in PCT/EP2016/076603 for X═NH.

Preparation of the New Cryptophycin Payloads of Formula (IV) forY═NH—CH₂

Preparation of the New Cryptophycin Payloads Bearing a Spacer withImproved Hydrophilicity

Without PABA Moiety

Step (i): peptidic coupling between the cryptophycin derivative and thedipeptide using coupling reagents such as, for example, EDC and HOBt;

Step (ii): deprotection of the Fmoc amine in the presence of a base suchas, for example, piperidine;

Step (iii): peptidic coupling with spacer 1 using coupling reagents suchas, for example, EDC and HOBt, or coupling with spacer 2 in the presenceof a base such as, for example, DIEA;

Step (iv): deprotection of the allyl ester in the presence of a catalystsuch as, for example, tetrakis-(triphenylphosphine)palladium;

Step (v): activation of the carboxylic acid as a NHS ester by treatmentwith DSC in the presence of a base such as, for example, DIEA.

Alternatively, new cryptophycin payloads bearing a spacer with improvedhydrophilicity may also be prepared as depicted in Scheme 9.

Step (i): activation of the carboxylic acid of linker 1 as a NHS esterby treatment with DSC in the presence of a base such as, for example,DIEA and coupling with the cryptophycin amine.

Step (ii): deprotection of the allyl ester in the presence of a catalystsuch as, for example, tetrakis-(triphenylphosphine)palladium;

Step (iii): activation of the carboxylic acid as a NHS ester bytreatment with DSC in the presence of a base such as, for example, DIEA.

Schemes 8 and 9 depicted the synthesis of payloads using the p-benzylicamine of C52 but may also apply to other cryptophycin compounds; itdepicted the synthesis using Val-Ala dipeptides but may also apply toother dipeptides; it depicted the synthesis using spacers 1 or 2 but mayalso apply to spacers 3 or 4.

-   -   With PABA Moeity

Step (i): peptidic coupling between Fmoc-Val-Ala-OH and 4-aminobenzylalcohol in the presence of a coupling reagent such as, for example,EEDQ;

Step (ii): activation of the benzylic alcohol as a p-nitrophenylcarbonate by treatment with p-nitrophenyl-chloroformate in the presenceof a base such as, for example, DIEA;

Step (iii): formation of a carbamate between the activated alcohol andthe cryptophycin amine in the presence of a base such as, for example,DIEA and deprotection of the Fmoc amine in the presence of a base suchas, for example, piperidine;

Step (iv): peptidic coupling with spacer 1 using coupling reagents suchas, for example, EDC and HOBt, or coupling with spacer 2 in the presenceof a base such as, for example, DIEA;

Step (v): deprotection of the allyl ester in the presence of a catalystsuch as, for example, tetrakis-(triphenylphosphine)palladium;

Step (vi): activation of the carboxylic acid as a NHS ester by treatmentwith DSC in the presence of a base such as, for example, DIEA.

Scheme 10 depicted the synthesis of payloads using Val-Ala dipeptidesbut may also apply to other dipeptides; it depicted the synthesis usingp-aminobenzyl alcohol (CAS number [623-04-1]) but may also apply toother aminobenzyl alcohol compounds which are commercially availablesuch as, for example, 4-(1-hydroxyethyl)-aniline (racemic (CAS number[14572-89-5]) or enantiopure (R) (CAS number [210754-25-9]) or (S) (CASnumber [500229-84-5])), 4-amino-α,α-dimethyl-benzene-methanol (CASnumber [23243-04-1]), 4-amino-α-methoxy-α-methyl-benzenemethanol (CASnumber [1379318-81-6]),4-amino-α-methyl-α-trifluoromethyl-benzenemethanol (CAS number[851652-56-7]), 2-amino-benzenemethanol (CAS number [5344-90-1]),2-amino-α-methyl-benzenemethanol (racemic (CAS number [10517-50-7]) orenantiopure (R) (CAS number [3205-21-8]) or (S) (CAS number[3205-21-8])), 6-amino-3-pyridinemethanol (CAS number [113293-71-3]),6-amino-α-methyl-3-pyridinemethanol (CAS number [1335054-83-5]),6-amino-α-ethyl-3-pyridinemethanol (CAS number [1355225-85-2]),6-amino-α,α-dimethyl-3-pyridinemethanol (CAS number [843646-03-8]),5-amino-3-pyridinemethanol (CAS number [873651-92-4]),2-amino-3-pyridinemethanol (CAS number [23612-57-9]),2-amino-α-methyl-3-pyridinemethanol (racemic (CAS number [869567-91-9])or enantiopure (R) (CAS number [936718-01-3]) or (S) (CAS number[936718-00-2])), 2-amino-α-ethyl-3-pyridinemethanol (CAS number[914223-90-8]), 2-amino-α,α-dimethyl-3-pyridinemethanol (CAS number[213666-96-7]), 3-amino-4-pyridinemethanol (CAS number [152398-05-5]),3-amino-α-methyl-4-pyridinemethanol (CAS number [1242470-88-7]),3-amino-α,α-methyl-4-pyridinemethanol (CAS number [13357-81-8]),4-amino-3-pyridinemethanol (CAS number [138116-34-4]),4-amino-α-methyl-3-pyridinemethanol (CAS number [741223-49-4]),4-amino-α,α-methyl-3-pyridinemethanol (CAS number [1339013-26-1]),3-amino-2-pyridinemethanol (CAS number [52378-63-9]),3-amino-α-methyl-2-pyridinemethanol (CAS number [954240-54-1]),3-amino-α,α-methyl-2-pyridinemethanol (CAS number [899438-57-4]); itdepicted the synthesis using the p-benzylic amine of C52 but may alsoapply to other cryptophycin compounds; it depicted the synthesis usingspacers 1 or 2 but may also apply to spacers 3 or 4.

Preparation of the New Cryptophycin Payloads of Formula (IV) Bearing aDipeptide with Improved Hydrophilicity

Without PABA Moiety

Step (i): peptidic coupling between the cryptophycin derivative and thedipeptide using coupling reagents such as, for example, EDC and HOBt anddeprotection of the Fmoc amine in the presence of a base such as, forexample, piperidine;

Step (ii): coupling to glutaric anhydride; one additional deprotectionstep of the side chain of substituted amino acid may be required priorto step (iii);

Step (iii): activation of the carboxylic acid as a NHS ester bytreatment with DSC in the presence of a base such as, for example, DIEA.

Scheme 11 depicted the synthesis of payloads using the p-benzylic amineof C52 but may also apply to other cryptophycin compounds; it depictedthe synthesis using dipeptide 1 but may also apply to dipeptide 5; itdepicted the synthesis using glutaric anhydride but may also apply tosuccinic anhydride or alkyl diacids which are commercially available forn ranging from 3 to 10.

With PABA Moiety

Step (i): formation of a carbamate between the activated alcohol and thecryptophycin amine in the presence of a base such as, for example, DIEAand deprotection of the Fmoc amine in the presence of a base such as,for example, piperidine;

Step (ii): coupling to glutaric anhydride; one additional deprotectionstep of the side chain of substituted amino acid may be required priorto step (iii);

Step (iii): activation of the carboxylic acid as a NHS ester bytreatment with DSC in the presence of a base such as, for example, DIEA.

Scheme 12 depicted the synthesis of payloads using the p-benzylic amineof C52 but may also apply to other cryptophycin compounds; it depictedthe synthesis using dipeptide 6 but may also apply to dipeptide 2.

Preparation of the New Cryptophycin Payloads of Formula (IV) BearingBoth a Spacer and a Dipeptide with Improved Hydrophilicity

Without PABA Moeity

Step (i): peptidic coupling between the cryptophycin derivative and thedipeptide using coupling reagents such as, for example, EDC and HOBt anddeprotection of the Fmoc amine in the presence of a base such as, forexample, piperidine;

Step (ii): peptidic coupling with spacer 3 using coupling reagents suchas, for example, EDC and HOBt, or coupling with spacer 4 in the presenceof a base such as, for example, DIEA;

Step (iii): deprotection of the allyl ester in the presence of acatalyst such as, for example, tetrakis-(triphenylphosphine)palladium;one additional deprotection step of the side chain of substituted aminoacid may be required prior to step (iv);

Step (iv): activation of the carboxylic acid as a NHS ester by treatmentwith DSC in the presence of a base such as, for example, DIEA.

Scheme 13 depicted the synthesis of payloads using the p-benzylic amineof C52 but may also apply to other cryptophycin compounds; it depictedthe synthesis using dipeptide 5 but may also apply to dipeptide 1; itdepicted the synthesis using spacers 3 or 4 but may also apply tospacers 1 or 2.

With PABA Moiety

Step (i): formation of a carbamate between the activated alcohol and thecryptophycin amine in the presence of a base such as, for example, DIEAand deprotection of the Fmoc amine in the presence of a base such as,for example, piperidine;

Step (ii): peptidic coupling with spacer 3 using coupling reagents suchas, for example, EDC and HOBt, or coupling with spacer 4 in the presenceof a base such as, for example, DIEA;

Step (iii): deprotection of the allyl ester in the presence of acatalyst such as, for example, tetrakis-(triphenylphosphine)palladium;one additional deprotection step of the side chain of substituted aminoacid may be required prior to step (iv);

Step (iv): activation of the carboxylic acid as a NHS ester by treatmentwith DSC in the presence of a base such as, for example, DIEA.

Scheme 14 depicted the synthesis of payloads using the p-benzylic amineof C52 but may also apply to other cryptophycin compounds; it depictedthe synthesis using dipeptide 6 but may also apply to dipeptide 2; itdepicted the synthesis using spacers 3 or 4 but may also apply tospacers 1 or 2.

Preparation of the New Cryptophycin Payloads of Formula (IV) for Y═O—CH2or S—CH2

Preparation of the New Cryptophycin Payloads Bearing a Spacer withImproved Hydrophilicity

Without PABA Moiety

Step (i): formation of a carbamate between the cryptophycin activatedalcohol and the amine in the presence of a base such as, for example,DIEA;

Step (ii): deprotection of the Fmoc amine in the presence of a base suchas, for example, piperidine;

Step (iii): peptidic coupling with spacer 1 using coupling reagents suchas, for example, EDC and HOBt, or coupling with spacer 2 in the presenceof a base such as, for example, DIEA;

Step (iv): deprotection of the allyl ester in the presence of a catalystsuch as, for example, tetrakis-(triphenylphosphine)palladium;

Step (v): activation of the carboxylic acid as a NHS ester by treatmentwith DSC in the presence of a base such as, for example, DIEA.

Scheme 15 depicted the synthesis of payloads using the activatedp-benzylic alcohol of C52 but may also apply to other cryptophycincompounds; it depicted the synthesis using Val-Ala dipeptides but mayalso apply to other dipeptides; it depicted the synthesis usingBoc-monoprotected ethylenediamine but may also apply to otherBoc-monoprotected diamines which are commercially available for nranging from 3 to 10; it depicted the synthesis using spacers 1 or 2 butmay also apply to spacers 3 or 4.

Alternatively, new cryptophycin payloads of formula (IV) bearing aspacer with improved hydrophilicity may also be prepared as depicted inScheme 16.

Step (i): formation of a carbamate between the cryptophycin activatedalcohol and linker 2 in the presence of a base such as, for example,DIEA;

Step (ii): deprotection of the allyl ester in the presence of a catalystsuch as, for example, tetrakis-(triphenylphosphine)palladium;

Step (iii): activation of the carboxylic acid as a NHS ester bytreatment with DSC in the presence of a base such as, for example, DIEA.

Scheme 16 depicted the synthesis of payloads using the activatedp-benzylic alcohol of C52 but may also apply to other cryptophycincompounds.

With PABA Moeity

Step (i): peptidic coupling between Fmoc-Val-Ala-OH and 4-aminobenzylalcohol in the presence of a coupling reagent such as, for example,EEDQ;

Step (ii): activation of the benzylic alcohol as a p-nitrophenylcarbonate by treatment with p-nitrophenyl-chloroformate in the presenceof a base such as, for example, DIEA;

Step (iii): formation of a carbamate between the activated alcohol andBoc-monoprotected ethylene diamine in the presence of a base such as,for example, DIEA;

Step (iv): deprotection of the Boc amine using a solution of HCl (forexample solution in dioxane) or of TFA;

Step (v): formation of a carbamate between the cryptophycin activatedalcohol and the amine in the presence of a base such as, for example,DIEA and deprotection of the Fmoc amine in the presence of a base suchas, for example, piperidine;

Step (vi): peptidic coupling with spacer 1 using coupling reagents suchas, for example, EDC and HOBt, or coupling with spacer 2 in the presenceof a base such as, for example, DIEA;

Step (vii): deprotection of the allyl ester in the presence of acatalyst such as, for example, tetrakis-(triphenylphosphine)palladium;

Step (viii): activation of the carboxylic acid as a NHS ester bytreatment with DSC in the presence of a base such as, for example, DIEA.

Scheme 17 depicted the synthesis of payloads using Val-Ala dipeptidesbut may also apply to other dipeptides; it depicted the synthesis usingp-aminobenzyl alcohol (CAS number [623-04-1]) but may also apply toother aminobenzyl alcohol compounds which are commercially availablesuch as, for example, 4-(1-hydroxyethyl)-aniline (racemic (CAS number[14572-89-5]) or enantiopure (R) (CAS number [210754-25-9]) or (S) (CASnumber [500229-84-5])), 4-amino-α,α-dimethyl-benzene-methanol (CASnumber [23243-04-1]), 4-amino-α-methoxy-α-methyl-benzenemethanol (CASnumber [1379318-81-6]),4-amino-α-methyl-α-trifluoromethyl-benzenemethanol (CAS number[851652-56-7]), 2-amino-benzenemethanol (CAS number [5344-90-1]),2-amino-α-methyl-benzenemethanol (racemic (CAS number [10517-50-7]) orenantiopure (R) (CAS number [3205-21-8]) or (S) (CAS number[3205-21-8])), 6-amino-3-pyridinemethanol (CAS number [113293-71-3]),6-amino-α-methyl-3-pyridinemethanol (CAS number [1335054-83-5]),6-amino-α-ethyl-3-pyridinemethanol (CAS number [1355225-85-2]),6-amino-α,α-dimethyl-3-pyridinemethanol (CAS number [843646-03-8]),5-amino-3-pyridinemethanol (CAS number [873651-92-4]),2-amino-3-pyridinemethanol (CAS number [23612-57-9]),2-amino-α-methyl-3-pyridinemethanol (racemic (CAS number [869567-91-9])or enantiopure (R) (CAS number [936718-01-3]) or (S) (CAS number[936718-00-2])), 2-amino-α-ethyl-3-pyridinemethanol (CAS number[914223-90-8]), 2-amino-α,α-dimethyl-3-pyridinemethanol (CAS number[213666-96-7]), 3-amino-4-pyridinemethanol (CAS number [152398-05-5]),3-amino-α-methyl-4-pyridinemethanol (CAS number [1242470-88-7]),3-amino-α,α-methyl-4-pyridinemethanol (CAS number [13357-81-8]),4-amino-3-pyridinemethanol (CAS number [138116-34-4]),4-amino-α-methyl-3-pyridinemethanol (CAS number [741223-49-4]),4-amino-α,α-methyl-3-pyridinemethanol (CAS number [1339013-26-1]),3-amino-2-pyridinemethanol (CAS number [52378-63-9]),3-amino-α-methyl-2-pyridinemethanol (CAS number [954240-54-1]),3-amino-α,α-methyl-2-pyridinemethanol (CAS number [899438-57-4]); itdepicted the synthesis using Boc-monoprotected ethylenediamine but mayalso apply to other Boc-monoprotected diamines which are commerciallyavailable for n ranging from 3 to 10; it depicted the synthesis usingthe activated p-benzylic alcohol of C52 but may also apply to othercryptophycin compounds; it depicted the synthesis using spacers 1 or 2but may also apply to spacers 3 or 4.

Preparation of the New Cryptophycin Payloads of Formula (IV) Bearing aDipeptide with Improved Hydrophilicity

Without PABA Moiety

Step (i): formation of a carbamate between the cryptophycin activatedalcohol and the dipeptide in the presence of a base such as, forexample, DIEA and deprotection of the Fmoc amine in the presence of abase such as, for example, piperidine;

Step (ii): coupling with glutaric anhydride; one additional deprotectionstep of the side chain of substituted amino acid may be required priorto step (iii)

Step (iii): activation of the carboxylic acid as a NHS ester bytreatment with DSC in the presence of a base such as, for example, DIEA.

Scheme 18 depicted the synthesis of payloads using the activatedp-benzylic alcohol of C52 but may also apply to other cryptophycincompounds; it depicted the synthesis using dipeptide 7 but may alsoapply to dipeptide 3; it depicted the synthesis using glutaric anhydridebut may also apply to succinic anhydride or alkyl diacids which arecommercially available for n ranging from 3 to 10.

With PABA Moiety

Step (i): formation of a carbamate between the cryptophycin activatedalcohol and the dipeptide in the presence of a base such as, forexample, DIEA and deprotection of the Fmoc amine in the presence of abase such as, for example, piperidine;

Step (ii): coupling with glutaric anhydride; one additional deprotectionstep of the side chain of substituted amino acid may be required priorto step (iii)

Step (iii): activation of the carboxylic acid as a NHS ester bytreatment with DSC in the presence of a base such as, for example, DIEA.

Scheme 19 depicted the synthesis of payloads using the activatedp-benzylic alcohol of C52 but may also apply to other cryptophycincompounds; it depicted the synthesis using dipeptide 8 but may alsoapply to dipeptide 4; it depicted the synthesis using glutaric anhydridebut may also apply to succinic anhydride or alkyl diacids which arecommercially available for n ranging from 3 to 10.

Preparation of the New Cryptophycin Payloads of Formula (IV) BearingBoth a Spacer and a Dipeptide with Improved Hydrophilicity

Without PABA Moeity

Step (i): formation of a carbamate between the cryptophycin activatedalcohol and the dipeptide in the presence of a base such as, forexample, DIEA and deprotection of the Fmoc amine in the presence of abase such as, for example, piperidine;

Step (ii): peptidic coupling with spacer 3 using coupling reagents suchas, for example, EDC and HOBt, or coupling with spacer 4 in the presenceof a base such as, for example, DIEA;

Step (iii): deprotection of the allyl ester in the presence of acatalyst such as, for example, tetrakis-(triphenylphosphine)palladium;one additional deprotection step of the side chain of substituted aminoacid may be required prior to step (iv)

Step (iv): activation of the carboxylic acid as a NHS ester by treatmentwith DSC in the presence of a base such as, for example, DIEA.

Scheme 20 depicted the synthesis of payloads using the p-benzylic amineof C52 but may also apply to other cryptophycin compounds; it depictedthe synthesis using dipeptide 7 but may also apply to dipeptide 3; itdepicted the synthesis using spacers 3 or 4 but may also apply tospacers 1 or 2.

With PABA Moiety

Step (i): formation of a carbamate between the cryptophycin activatedalcohol and the dipeptide in the presence of a base such as, forexample, DIEA and deprotection of the Fmoc amine in the presence of abase such as, for example, piperidine;

Step (ii): peptidic coupling with spacer 3 using coupling reagents suchas, for example, EDC and HOBt, or coupling with spacer 4 in the presenceof a base such as, for example, DIEA;

Step (iii): deprotection of the allyl ester in the presence of acatalyst such as, for example, tetrakis-(triphenylphosphine)palladium;one additional deprotection step of the side chain of substituted aminoacid may be required prior to step (iv);

Step (iv): activation of the carboxylic acid as a NHS ester by treatmentwith DSC in the presence of a base such as, for example, DIEA.

Scheme 21 depicted the synthesis of payloads using the p-benzylic amineof C52 but may also apply to other cryptophycin compounds; it depictedthe synthesis using dipeptide 8 but may also apply to dipeptide 4; itdepicted the synthesis using spacers 3 or 4 but may also apply tospacers 1 or 2.

Preparation of the Conjugates of Formula (V)

The conjugates of formula (V) of the present invention can obtained viathe process comprising at least the steps of:

(i) placing in contact and leaving to react:

-   -   an optionally buffered aqueous solution of an antibody,        optionally modified by means of a modifying agent, and    -   a solution of a cryptophycin payload of formula (IV) as defined        in the present invention,

the chemical group RCG1 of the cryptophycin payload of formula (IV)being reactive towards the chemical groups RCG2 present on thepolypeptide such as the antibody especially towards the amino groupspresent on antibodies, the said chemical groups RCG2 having beenintroduced, where appropriate, by the modifying agent, so as to attachthe cryptophycin payload of formula (IV) to the antibody by formation ofa covalent bond;

(ii) and then optionally to separate the conjugate of formula (V) formedin step (i) from the cryptophycin payload of formula (IV) and/or fromthe unreacted antibody and/or from any aggregates that may have formed.

According to one variant for example, in step (ii) the conjugate offormula (V) from step (i) is separated only from the unreactedcryptophycin payload of formula (IV) and from any aggregates formed, andany unreacted antibody is left in the solution.

The function of the placing in contact is to react the chemical groupsRCG1 and RCG2 in order to ensure attachment of the cryptophycin payloadof formula (IV) to the polypeptide such as the antibody by formation ofa covalent bond; such as,

-   -   when RCG1 represents R_(a)Z_(a)—C(═O), the reaction preferably        takes place on the amino functions of the antibody, such as the        ε-amino groups borne by the side chains of the lysine (Lys)        residues of the antibody and the α-amino groups of N-terminal        amino acids of antibody heavy and light chains. A conjugate of        the following formula is obtained in this case:        mAb-[NH—C(═O)-L*-Y-Crypto]_(d) with L* representing a fragment        of a linker L comprising as RCG1 a R_(a)Z_(a)—C(═O) group and d        representing the drug-to-antibody ratio or DAR;    -   when RCG1 represents a chlorine atom or a maleimido or        haloacetamido group, the antibody may comprise thiol chemical        groups;    -   when RCG1 represents an azido group, the antibody may comprise a        C≡CH moiety or an activated triple bond such as a cyclooctyne        group;    -   when RCG1 represents NH₂ group, the reaction may take place on        amide function of the antibody using an enzymatic catalysis,        such as the amide groups borne by the side chains of glutamine        (Gln) residues of an antibody. A conjugate of the following        formula is obtained in this case: mAb-[C(═O)—NH-L*-Crypto]_(d)        with L* representing a fragment of a linker L comprising as RCG1        a NH₂ group and such that L representing a L*NH₂ group and d        representing the drug-to-antibody ratio or DAR;    -   when RCG1 represents a C≡CH group or an activated C≡C group such        as a cyclooctyne moiety, the antibody may comprises azido        groups.

The term “aggregates” means associations that may form between two ormore antibodies, the antibodies possibly having been modified byconjugation. Aggregates are liable to form under the influence of a widevariety of parameters such as a high concentration of antibody in thesolution, the pH of the solution, high shear forces, the number ofgrafted drugs and their hydrophobic nature, the temperature (see thereferences cited in the introduction of J. Membrane Sci. 2008, 318,311-316), the influence of some of them, however, having not beenclearly elucidated. In the case of proteins or antibodies, reference maybe made to AAPS Journal, “Protein Aggregation and Bioprocessing” 2006,8(3), E572-E579. The aggregate content may be determined via knowntechniques such as SEC (see in this respect Analytical Biochemistry1993, 212 (2), 469-480).

The aqueous solution of the antibody may be buffered with buffers forexample, potassium phosphate or HEPES or a mixture of buffers such asbuffer A described later. The buffer depends on the nature of theantibody. The cryptophycin payload of formula (IV) is dissolved in apolar organic solvent such as DMSO or DMA.

The reaction takes place at a temperature generally ranging from 20° C.to 40° C. The reaction time may be ranging from 1 to 24 hours. Thereaction between the antibody and the cryptophycin payload of formula(IV) may be monitored by SEC with a refractometric and/or ultravioletdetector and/or HRMS in order to determine its degree of progress. Ifthe degree of substitution is insufficient, the reaction can be left forlonger and/or cryptophycin compound can be added. Reference may be madeto the example section for further details regarding particularconditions. Particular embodiments are described in Examples 3, 6, 16,19, 23, 26, 29, 32, 35 and 41.

A person skilled in the art has at his disposal various chromatographictechniques for the separation of step (ii): the conjugate may bepurified, for example, by steric exclusion chromatography (SEC), byadsorption chromatography (for instance ion exchange, IEC), byhydrophobic interaction chromatography (HIC), by affinitychromatography, by chromatography on mixed supports such as ceramichydroxyapatite, or by HPLC. Purification by dialysis or diafiltrationmay also be used.

After step (i) or (ii), the solution of the conjugate may undergo anultrafiltration and/or diafiltration step (iii). After these steps, theconjugate in aqueous solution is thus obtained.

Antibody

The antibody can be a monoclonal antibody selected from the groupconsisting of a murine, chimeric, a humanized and a human antibody.

In one embodiment, the antibody is a monospecific antibody, i.e. anantibody specifically binding to one single target. Alternatively, itmight be a multispecific antibody.

In one embodiment, the antibody is a IgG antibody, for instance an IgG₁,an IgG₂, an IgG₃ or an IgG₄ antibody.

The antibody according to the invention specifically binds to a target,thereby directing the biologically active compound as a cytotoxiccompound towards said target. As used herein, “specifically binds” or“binds specifically to” or “binds to” or the like, means that anantibody or antigen-binding fragment thereof forms a complex with anantigen that is relatively stable under physiological conditions.Specific binding can be characterized by an equilibrium dissociationconstant (K_(D)) of at least about 1×10⁻⁸ M or less (e.g., a smallerK_(D) denotes a tighter binding). Methods for determining whether twomolecules specifically bind are well known in the art and include, forexample, equilibrium dialysis, surface plasmon resonance, and the like.

As described herein, antibodies have been characterized, for example, bytheir specific binding to target and/or target antigen using surfaceplasmon resonance, e.g., BIACORE™.

The target typically corresponds to a protein expressed at the cellsurface, e.g. a protein expressed at the surface of tumour cells.

In one embodiment, the target is the EphA2 receptor. The EphA2 receptoris an Ephrin receptor, and is also referred to as “Eph receptor A₂” or“Epithelial Cell Receptor Protein-Tyrosine kinase”. The antibodyspecifically binding to the EphA2 receptor might for instance correspondto one of the antibodies described in WO2008/010101 or WO2011/039724.

The antibody may optionally be modified with a modifying agent so as topromote the attachment of the cryptophycin payload as previouslydescribed. The antibody may especially be monoclonal, polyclonal ormultispecific. It may also be an antibody fragment. It may also be amurine, human, humanized or chimeric antibody. The antibody used in theexamples of the present invention is hu2H11_R3574, an antagonistantibody against EphA2 receptor. The sequence of hu2H11_R3574 isrepresented by SEQ ID NO: 1 (light chain of antibody hu2H11_R35R74) andby SEQ ID NO:2 (heavy chain of antibody hu2H11_R35R74) which correspondto respectively SEQ ID NO: 16 and SEQ ID NO:18 represented inWO2011039724 A1.

Conjugate

A conjugate generally comprises from about 1 to 10 cryptophycincompounds covalently attached to the antibody (this is the degree ofgrafting or “drug-to-antibody ratio” or “DAR”). This number varies as afunction of the nature of the antibody and of the cryptophycin compound,and also of the operating conditions used in the conjugation process(for example the number of equivalents of cryptophycin compound relativeto the antibody, the reaction time, the nature of the solvent and of anycosolvent). Placing of the antibody and the cryptophycin compound incontact leads to a mixture comprising several conjugates that areindividually distinguished from each other by different DARs; optionallythe unreacted antibody; optionally aggregates. The DAR that isdetermined on the final solution thus corresponds to an average DAR. TheDAR may be calculated from the deconvolution of the SEC-HRMS spectrum ofthe conjugate. The DAR (HRMS) is for example greater than 0.5, forinstance ranging from 1 to 10, such as ranging from 2 to 7.

The conjugate may be used as an anticancer agent. Owing to the presenceof the antibody, the conjugate is made highly selective towards tumorcells rather than healthy cells. This makes it possible to direct thecryptophycin compound in an environment similar thereto or directlytherein. It is possible to treat solid or liquid cancers. The conjugatemay be used alone or in combination with at least one other anticanceragent.

The conjugate is formulated in the form of a buffered aqueous solutionat a concentration generally ranging from 1 to 10 mg/mL. This solutionmay be injected in perfusion form per se or may be rediluted to form aperfusion solution.

EXAMPLES

The examples which follow describe the preparation of certain compoundsin accordance with the invention. These examples are not limitative, andmerely illustrate the present invention.

Analytical Methods Used

High Pressure Liquid Chromatoqraphy-Mass Spectrometry (LCMS)

Method A

Spectra have been obtained on a Waters UPLC-SQD system in positiveand/or negative electrospray mode (ES+/−). Chromatographic conditionswere the following: Column: ACQUITY BEH C18—1.7 μm—2.1×50 mm; solvents:A: H₂O (0.1% formic acid), B: CH₃CN (0.1% formic acid); columntemperature: 50° C.; flow rate: 0.8 mL/min; gradient (2.5 min): from 5to 100% of B in 1.8 min; 2.4 min: 100% of B; 2.45 min: from 100 to 5% ofB in 0.05 min.

Method B

Spectra have been obtained on a Waters UPLC-SQD system in positiveand/or negative electrospray mode (ES+/−). Chromatographic conditionswere the following: Column: ACQUITY BEH C18—1.7 μm—2.1×50 mm; solvents:A: H₂O (0.1% formic acid), B: CH₃CN (0.1% formic acid); columntemperature: 50° C.; flow rate: 0.6 mL/min; gradient (2 min): from 5 to50% of B in 1 min; from 50 to 100% of B in 0.3 min; 100% of B during0.15 min; from 100 to 5% de B in 0.3 min and 5% of B during 0.25 min.

Method C

Spectra have been obtained on a Waters UPLC-SQD system in positiveand/or negative electrospray mode (ES+/−). Chromatographic conditionswere the following: Column: ACQUITY BEH C18—1.7 μm—2.1×50 mm; solvents:A: H₂O (0.1% formic acid), B: CH₃CN (0.1% formic acid); columntemperature: 50° C.; flow rate: 0.8 mL/min; gradient (5 min): from 5 to100% of B in 4.2 min; 4.6 min: 100% of B; 4.8 min: 5% of B.

Method D

Spectra have been obtained on a Waters XeVo-Qtof system in positiveelectrospray mode (ES+). Chromatographic conditions were the following:Column: ACQUITY BEH C18—1.7 μm—2.1×100 mm; solvents: A: H₂O (0.1% formicacid), B: CH₃CN (0.1% formic acid); column temperature: 45° C.; flowrate: 0.6 mL/min; gradient (5.3 min): 5% of B from 0 to 0.3 min, 4 min:100% of B; 4.6 min: 100% of B; 5.3 min: 5% of B.

Method E

Spectra have been obtained on a Waters UPLC-SQD system in positiveand/or negative electrospray mode (ES+/−). Chromatographic conditionswere the following: Column: ACQUITY BEH C18—1.7 μm—2.1×50 mm; solvents:A: H₂O (0.1% formic acid), B: CH₃CN (0.1% formic acid); columntemperature: 45° C.; flow rate: 0.8 mL/min; gradient (10 min): from 5 to100% of B in 8.6 min; 9.6 min: 100% of B; 9.8 min: 5% of B.

¹H Nuclear Magnetic Resonance (NMR)

The ¹H NMR spectra were acquired on a Bruker Avance spectrometer, eitherof model DRX-300, DRX-400 or DRX-500. The chemical shifts (6) are givenin ppm.

Size Exclusion Chromatography-High Resolution Mass Spectrometry(SEC-HRMS)

The chromatographic analysis was performed on an Agilent HP1100 machineand a Waters BEH SEC 200 1.7 μm (2.1×150 mm) column at 30° C. with aflow rate of 0.5 mL/min and an isocratic elution of (A) 25 mM ammoniumformate+1% formic acid/(B) CH3CN+0.1% formic acid 70/30 for 15 minutes.The mass spectrometry was performed on a Waters QTOF-II machine withelectrospray ionization in positive mode (ES+). The mass spectra weredeconvoluted with the Waters MaxEnt1 software.

Analytical Size Exclusion Chromatography (SEC)

The analysis was performed on a Waters Alliance HPLC system or a HitachiLachrom HPLC system equipped with a photodiode array detector and aTosoh Bioscience TSKgel G3000 SWXL 5 μm column (7.8×300 mm) with a flowrate of 0.5 mL/min and an isocratic elution of 30 minutes with a pH 7buffer containing 0.2 M of KCl, 0.052 M of KH₂PO₄, 0.107 M of K₂HPO₄ and20% by volume of isopropanol.

Buffers

-   -   Buffer A (pH 6.5): NaCl (50 mM), potassium phosphate buffer (50        mM), EDTA (2 mM)    -   Buffer B (pH 6.5): NaCl (140 mM), potassium and sodium phosphate        (9.6 mM)    -   DPBS (pH 7.2): KCl (2.7 mM), NaCl (137 mM), KH₂PO₄ (1.47 mM),        Na₂HPO₄ (8.10 mM)    -   PBS (pH 7.4): KH₂PO₄ (1.06 mM), NaCl (155.17 mM), Na₂HPO₄-7H₂O        (2.97 mM)

General Method Used for the Preparation of Antibody-Drug Conjugate (ADC)

A solution of antibody in an aqueous buffer composed of a 96:4 mixtureof buffer A and 1 N HEPES was treated with an excess (5 to 10equivalents) of a solution at approximatively 10 mM of cryptophycinpayload in DMA such that the final antibody concentration is 3 mg/mL andthe percentage of DMA in the aqueous buffer is 20%. After stirring for 1to 4 hours, the mixture was analysed by SEC-HRMS to determine the DAR onthe population of monomeric antibodies. If the DAR was foundinsufficient (<3.5-4), the mixture was treated with a further excess (1to 5 equivalents) of cryptophycin solution in DMA for 2 to 4 additionalhours at RT under stirring. The mixture was purified by gel filtrationusing a Superdex 200 pg matrix (HiLoad 16/60 or 26/60 desalting column,GEHealthcare) pre-equilibrated in aqueous buffer pH 6.5 (buffer B orDPBS) containing 10 to 20% of NMP or a Sephadex™ G25 matrix (Hiprep26/10 desalting column, GEHealthcare) pre-equilibrated in aqueous bufferpH 6.5 (buffer B or DPBS) containing 5 to 10% of NMP. The fractionscontaining the monomeric conjugated antibody were pooled andconcentrated on Amicon Ultra-15 (10k or 50k Ultracel membrane,Millipore) to a concentration of between 2 and 5 mg/mL. A bufferexchange or a dilution in the appropriate buffer was then performed toformulate the conjugate in the final buffer. In the case of a bufferexchange, it was realized by gel filtration using a Sephadex™ G25 matrix(NAP-5, NAP-10, NAP-25/PD-10 or Hiprep 26/10 desalting columns,GEHealthcare) pre-equilibrated with the final aqueous buffer whosecomposition and pH are suited to each conjugate. The conjugate wasfinally filtered through a Steriflip® filter unit (0.22 μm Durapore®PVDF membrane, Millipore). The final conjugate was assayed by UVspectrometry or SEC-HPLC so as to measure the conjugate concentration,by SEC-HPLC so as to determine the monomeric purity and by SEC-HRMS soas to determine the DAR from the deconvolution of the mass spectrum ofthe conjugate.

Synthesis of Examples 1 to 3: PEG4-Val-Ala-C52 Benzylic Amine, NHS Esterof PEG4-Val-Ala-C52 Benzylic Amine and Corresponding ADC

Compound 1: allyl 3-((tert-butoxycarbonyl)amino)propanoate

To a solution of Boc-β-Ala-OH (CAS number [3303-84-2], 2 g, 10.04 mmol)in DMF (50 mL) were added cesium carbonate (6.88 g, 21.09 mmol) andallyl bromide (965.52 μL, 11.05 mmol). The reaction medium was stirredat RT overnight. At this time, the reaction medium was concentrated invacuo, then diluted with toluene and concentrated in vacuo severaltimes. The crude product was diluted with water (100 mL) and extractedwith EtOAc (3×300 mL). The combined organic phases were washed withbrine, dried over MgSO₄, filtered, concentrated in vacuo and purified byflash chromatography on 90 g of silica gel (gradient elution DCM/MeOH)to give 1.5 g of compound 1 (65%).

RMN ¹H (400 MHz, 6 in ppm, DMSO-d6): 1.37 (s, 9H); 2.45 (t, J=6.9 Hz,2H); 3.17 (q, J=6.9 Hz, 2H); 4.54 (m, 2H); 5.20 (qd, J=1.5 and 10.6 Hz,1H); 5.29 (qd, J=1.9 and 17.3 Hz, 1H); 5.91 (m, 1H); 6.85 (broad t,J=6.9 Hz, 1H).

Compound 2: allyl 3-aminopropanoate 2,2,2-trifluoroacetate

To a solution of compound 1 (1.5 g, 6.54 mmol) in DCM (30 mL) was addedTFA (5.09 mL, 65.42 mmol). The reaction medium was stirred at RTovernight. At this time, the reaction medium was concentrated in vacuo,then diluted with toluene and concentrated in vacuo twice to afford 1.6g of compound 2 (quant.).

Compound 3: allyl 3-((3-(tert-butoxy)-3-oxopropyl)amino)propanoate

To a solution of compound 3 (1.6 g, 6.58 mmol) in DMSO (15 mL) wereadded DIEA (2.30 mL, 13.16 mmol) and tert-butyl acrylate (973.49 μL,6.58 mmol). The reaction medium was heated 1 h at 60° C. usingmicrowaves. At this time, the reaction medium was diluted with H₂O (10mL) and extracted with EtOAc. The combined organic phases were washedwith brine, dried over MgSO₄, filtered, concentrated in vacuo andpurified by flash chromatography on 80 g of silica gel (gradient elutionDCM/MeOH) to give 678 mg of compound 3 (40%).

RMN ¹H (400 MHz, δ in ppm, DMSO-d6): 1.39 (s, 9H); 1.73 (broad m, 1H);2.29 (t, J=6.9 Hz, 2H); 2.44 (t, J=6.9 Hz, 2H); 2.68 (t, J=6.9 Hz, 2H);2.73 (t, J=6.9 Hz, 2H); 4.53 (m, 2H); 5.20 (qd, J=1.5 and 10.6 Hz, 1H);5.30 (qd, J=1.9 and 17.3 Hz, 1H); 5.91 (m, 1H).

Compound 4: tert-butyl 2,5,8,11-tetraoxatetradecan-14-oate

Under argon, a mixture of triethylene glycol monomethyl ether (CASnumber [112-35-6], 3 g, 17.36 mmol) and sodium (3.99 mg, 173.57 μmol) inTHF (9 mL) was stirred for 2 h at 50° C. then overnight at RT. At thistime, tert-butyl acrylate (3.08 ml, 20.83 mmol) was added, stirred for 2h at 40° C. then overnight at RT. After that, the reaction medium wasconcentrated in vacuo, then diluted H₂O and extrated with EtOAc. Thecombined organic phases were washed with brine, dried over MgSO₄,filtered, concentrated in vacuo and purified by flash chromatography on90 g of silica gel (gradient elution heptane/EtOAc) to give 3.3 g ofcompound 4 (65%).

Compound 5: 2,5,8,11-tetraoxatetradecan-14-oic Acid

To a solution of compound 4 (2.5 g, 8.55 mmol) in DCM (30 mL) was addedTFA (6.65 mL, 85.51 mmol). The reaction medium was stirred overnight atRT. At this time, the reaction medium was concentrated in vacuo, thendiluted with toluene and concentrated in vacuo three times to give 3 gof compound 5 (quant.).

Compound 6: 2,5,8,11-tetraoxatetradecan-14-oyl chloride

To compound 5 (800 mg, 3.39 mmol) was added thionyl chloride (3.68 mL,50.79 mmol). The reaction medium was heated 3 h at 60° C. After cooling,the reaction medium was concentrated in vacuo, diluted with DCM andconcentrated in vacuo twice to give 870 mg of compound 6 (quant.).

Compound 7: allyl15-(3-(tert-butoxy)-3-oxopropyl)-14-oxo-2,5,8,11-tetraoxa-15-azaoctadecan-18-oate

At 0° C., under argon, to a solution of compound 3 (678 mg, 2.63 mmol)and DIEA (533.21 μL, 3.16 mmol) in DCM (10 mL) was added a solution ofcompound 6 (738.21 mg, 2.90 mmol) in DCM (3 mL). After 2 h, the reactionmedium was washed with H₂O (10 mL) then brine, dried over MgSO₄,filtered, concentrated in vacuo and purified by flash chromatography on40 g of silica gel (gradient elution DCM/MeOH) to give 410 mg ofcompound 7 (33%).

RMN ¹H (400 MHz, δ in ppm, DMSO-d6): 50/50 conformer mixture; 1.39 (s,4.5H); 1.40 (s, 4.5H); 2.39 (t, J=7.2 Hz, 1H); 2.47 to 2.60 (partiallymasked m, 4H); 2.66 (t, J=7.2 Hz, 1H); 3.23 (s, 1.5H); 3.24 (s, 1.5H);3.38 to 3.65 (m, 18H); 4.54 (m, 2H); 5.19 to 5.34 (m, 2H); 5.85 to 5.97(m, 1H).

Compound 8:15-(3-(allyloxy)-3-oxopropyl)-14-oxo-2,5,8,11-tetraoxa-15-azaoctadecan-18-oicAcid

A solution of compound 7 (150 mg, 315.41 μmol) in DCM (3 mL) and TFA(236.66 μL, 3.15 mmol) was stirred at RT overnight. At this time, thereaction medium was concentrated in vacuo, diluted with toluene andconcentrated in vacuo three times to give 130 mg of compound 8 (98%).

Compound 9: allyl15-(3-((2,5-dioxopyrrolidin-1-yl)oxy)-3-oxopropyl)-14-oxo-2,5,8,11-tetraoxa-15-azaoctadecan-18-oate

To a solution of compound 8 (130 mg, 309.92 μmol) in THF (5 mL) wereadded DIEA (104.53 μL, 619.83 μmol) and DSC (97.21 mg, 371.90 μmol). Thereaction medium was stirred at RT overnight. At this time, the reactionmedium was concentrated in vacuo and purified by flash chromatography on4 g of silica gel (gradient elution DCM/iPrOH) to give 107 mg ofcompound 9 (67%).

Compound 10: (9H-fluoren-9-yl)methyl((2S)-1-(((2S)-1-((4-((2R,3S)-3-(1-((3S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13-en-16-yl)ethyl)oxiran-2-yl)benzyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate

To a solution of(3S,10R,16S,E)-16-((S)-1-((2R,3R)-3-(4-(aminomethyl)phenyl)-oxiran-2-yl)ethyl)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-1,4-dioxa-8,11-diazacyclohexadec-13-ene-2,5,9,12-tetraone(the synthesis of which was described in WO2011001052, compound 77, 150mg, 214.82 μmol) in DCM was added HOBt (31.93 mg, 236.31 μmol), EDC(41.83 μL, 236.31 μmol) and Fmoc-Val-Ala-OH (CAS number [150114-97-9],79.36 mg, 193.34 μmol). The reaction medium was stirred for 3 h at RT.Then H₂O (20 mL) was added and the mixture was extracted with DCM (2×15mL). The combined organic phases were washed with brine, dried overMgSO₄, filtered, concentrated in vacuo and purified by flashchromatography on 5 g of silica gel (gradient elution DCM/MeOH) to give200 mg of compound 10 (85%).

Compound 11:(2S)-2-amino-N-((2S)-1-((4-((2R)-3-((S)-1-((3S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13-en-16-yl)ethyl)oxiran-2-yl)benzyl)amino)-1-oxopropan-2-yl)-3-methylbutanamide

A mixture of compound 10 (200 mg, 183.37 μmol) in DCM (10 mL) andpiperidine (183.39 μL, 1.83 mmol) was stirred for 2 h at RT. Thereaction medium was concentrated in vacuo, solubilized in DCM beforeaddition of Et₂O. The precipitate was filtered, washed with Et₂O anddried to give 180 mg of crude that was purified by flash chromatographyon 10 g of amino-propyl modified silica gel (gradient elution DCM/MeOH)to give 136 mg of compound 11 (85%).

Compound 12: allyl15-(3-(((2S)-1-(((2S)-1-((4-((2R,3S)-3-(1-((3S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13-en-16-yl)ethyl)oxiran-2-yl)benzyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-oxopropyl)-14-oxo-2,5,8,11-tetraoxa-15-azaoctadecan-18-oate

To a solution of compound 11 (60 mg, 69.09 μmol) in THF (5 mL) wereadded compound 9 (42.82 mg, 82.91 μmol) and DIEA (17.48 μL, 103.63μmol). The reaction medium was stirred for 3 h at RT. At this time, itwas concentrated in vacuo and purified by flash chromatography on 10 gof silica gel (gradient elution DCM/MeOH) to give 60 mg of compound 12(68%).

RMN ¹H (MHz, δ in ppm, DMSO-d6): 60/40 conformer mixture; 0.78 (d, J=7.0Hz, 6H); 0.80 to 0.87 (m, 6H); 1.00 (s, 3H); 1.04 (d, J=7.0 Hz, 3H);1.12 (s, 3H); 1.24 (split d, J=7.0 Hz, 3H); 1.30 (m, 1H); 1.51 to 1.63(m, 2H); 1.75 to 1.83 (m, 1H); 1.95 (m, 1H); 2.26 (m, 1H); 2.30 to 2.73(m, 8H); 2.95 to 3.05 (m, 3H); 3.23 (s, 3H); 3.31 (masked m, 1H); 3.40to 3.64 (m, 18H); 3.81 (s, 3H); 3.87 (d, J=2.1 Hz, 1H); 4.12 to 4.33 (m,5H); 4.53 (m, 1.2H); 4.55 (m, 0.8H); 4.91 (m, 1H); 5.11 (m, 1H); 5.21(m, 1H); 5.28 (qd, J=1.9 and 17.3 Hz, 0.6H); 5.31 (qd, J=1.9 and 17.3Hz, 0.4H); 5.78 (split d, J=15.7 Hz, 1H); 5.85 to 5.95 (m, 1H); 6.47(ddd, J=4.2, 11.5 and 15.7 Hz, 1H); 7.05 (d, J=8.7 Hz, 1H); 7.17 (dd,J=2.3 and 8.7 Hz, 1H); 7.19 to 7.26 (m, 5H); 7.28 (d, J=2.3 Hz, 1H);7.94 (d, J=9.2 Hz, 0.4H); 8.04 (m, 1H); 8.10 (d, J=7.6 Hz, 0.6H); 8.32(m, 1H); 8.35 (d, J=8.2 Hz, 1H).

Example 1:15-(3-(((S)-1-(((S)-1-((4-((2R,3R)-3-((S)-1-((3S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13-en-16-yl)ethyl)oxiran-2-yl)benzyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-oxopropyl)-14-oxo-2,5,8,11-tetraoxa-15-azaoctadecan-18-oicAcid

Under argon, to a solution of compound 12 (60 mg, 47.25 μmol) in DCM (5mL) were added tetrakis(triphenylphosphine)palladium(0) (2.76 mg, 2.36μmol) and 1,3-dimethylbarbituric acid (22.58 mg, 141.74 μmol). Thereaction medium was stirred for 1 h at RT and concentrated in vacuo. Thecrude product was purified by flash chromatography on 4 g of silica gel(gradient elution DCM/MeOH) to give 31 mg of example 1 (53%).

RMN ¹H (500 MHz, δ in ppm, DMSO-d6): 0.79 (d, J=7.0 Hz, 6H); 0.80 to0.88 (m, 6H); 1.00 (s, 3H); 1.04 (d, J=7.0 Hz, 3H); 1.12 (s, 3H); 1.23(m, 3H); 1.31 (m, 1H); 1.51 to 1.62 (m, 2H); 1.80 (m, 1H); 1.95 (m, 1H);2.27 (m, 1H); 2.35 to 2.60 (partially masked m, 6H); 2.62 to 2.73 (m,2H); 2.93 to 3.06 (m, 3H); 3.23 (s, 3H); 3.28 to 3.35 (masked m, 1H);3.36 to 3.65 (m, 18H); 3.81 (s, 3H); 3.87 (d, J=2.0 Hz, 1H); 4.11 to4.34 (m, 5H); 4.91 (m, 1H); 5.10 (m, 1H); 5.79 (d, J=15.5 Hz, 1H); 6.48(ddd, J=3.8, 11.3 and 15.5 Hz, 1H); 7.05 (d, J=8.7 Hz, 1H); 7.17 (dd,J=2.0 and 8.7 Hz, 1H); 7.24 (m, 5H); 7.29 (d, J=2.0 Hz, 1H); 7.95 to8.18 (m, 2H); 8.30 to 8.46 (m, 2H); 12.27 (broad m, 1H). LCMS (A): ESm/z=615 [M+2H]²⁺; m/z=1227 [M−H]⁻; m/z=1229 [M+H]⁺; t_(R)=1.26 min.

Example 2: 2,5-dioxopyrrolidin-1-yl15-(3-(((S)-1-(((S)-1-((4-((2R,3R)-3-((S)-1-((3S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13-en-16-yl)ethyl)oxiran-2-yl)benzyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-oxopropyl)-14-oxo-2,5,8,11-tetraoxa-15-azaoctadecan-18-oate

To a solution of example 1 (30 mg, 24.39 μmol) in THF (5 mL) were addedDSC (7.65 mg, 29.27 μmol) and DIEA (9.88 μL, 58.54 μmol). The reactionmedium was stirred at RT overnight then concentrated in vacuo andpurified by flash chromatography on 5 g of diol modified silica gel(gradient elution DCM/iPrOH) to give 17 mg of example 2 (53%).

RMN ¹H (500 MHz, δ in ppm, DMSO-d6): 70/30 conformer mixture; 0.79 (d,J=7.0 Hz, 6H); 0.80 to 0.87 (m, 6H); 1.00 (s, 3H); 1.04 (d, J=7.0 Hz,3H); 1.12 (s, 3H); 1.21 to 1.34 (m, 4H); 1.51 to 1.62 (m, 2H); 1.80 (m,1H); 1.93 (m, 1H); 2.27 (m, 1H); 2.35 to 2.72 (partially masked m, 6H);2.81 (s large, 4H); 2.88 to 3.06 (m, 5H); 3.23 (s, 3H); 3.28 to 3.37(masked m, 1H); 3.40 to 3.79 (m, 18H); 3.81 (s, 3H); 3.88 (s, 1H); 4.13to 4.33 (m, 5H); 4.90 (m, 1H); 5.10 (m, 1H); 5.79 (d, J=15.5 Hz, 1H);6.46 (ddd, J=3.8, 11.5 and 15.5 Hz, 1H); 7.05 (d, J=8.7 Hz, 1H); 7.17(broad s, 1H); 7.24 (m, 5H); 7.29 (broad s, 1H); 7.94 (d, J=8.8 Hz,0.3H); 8.06 (m, 1H); 8.12 (d, J=8.1 Hz, 0.7H); 8.32 (m, 1H); 8.36 (d,J=8.1 Hz, 1H). LCMS (A): ES m/z=663.5 [M+2H]²⁺; m/z=1324 [M−H]⁻;m/z=1326 [M+H]⁺; m/z=1374 [M−H+HCO₂H]⁻; t_(R)=1.3 min.

Example 3: hu2H11_R35-74-Ex2

The general method described previously was used for the preparation ofexample 3. 15 mg of hu2H11_R35-74 were reacted with 55.2 μL of a 9.1 mMsolution of example 2 in DMA (10 eq.) for 3 h 30. After purification onSuperdex 200 pg in buffer B pH 6.5+10% NMP, concentration on AmiconUltra-15, buffer exchange on Nap-10 in buffer B pH 6.5+5% NMP andfiltration on 0.22 μm PVDF filter, 3.36 mg of example 3 were obtained asa colorless limpid solution at a concentration of 2.24 mg/mL with a DARof 3.4 (HRMS), a monomeric purity of 99.8% and a global yield of 22%.

SEC-HRMS: m/z=149354 (naked mAb); m/z=150563 (D1); m/z=151774 (D2);m/z=152966 (D3); m/z=154169 (D4); m/z=155410 (D5); m/z=156623 (D6);m/z=157835 (D7); m/z=159051 (D8).

Synthesis of Examples 4 to 6: sulfo-Val-Ala-C52 Benzylic Amine, NHSEster of sulfo-Val-Ala-C52 Benzylic Amine and Corresponding ADC

Compound 13:4-((3-(allyloxy)-3-oxopropyl)(3-(tert-butoxy)-3-oxopropyl)amino)-4-oxobutanoicAcid

A solution of compound 3 (958 mg, 3.72 mmol) in DCM (5 mL) and succinicanhydride (752.65 mg, 7.45 mmol) was stirred for 3 h at RT. The reactionmedium was concentrated in vacuo and purified by flash chromatography on24 g of silica gel (gradient elution DCM/MeOH) to give 1.3 g of compound13 (98%).

RMN ¹H (400 MHz, δ in ppm, DMSO-d6): 50/50 conformer mixture; 1.39 (s,4.5H); 1.40 (s, 4.5H); 2.33 to 2.43 (m, 3H); 2.45 to 2.55 (partiallymasked m, 4H); 2.69 (t, J=7.5 Hz, 1H); 3.40 (t, J=7.5 Hz, 1H); 3.45 (t,J=7.5 Hz, 1H); 3.53 (t, J=7.5 Hz, 1H); 3.59 (t, J=7.5 Hz, 1H); 4.53 (m,1H); 4.59 (m, 1H); 5.19 to 5.35 (m, 2H); 5.91 (m, 1H); 12.06 (broad m,1H).

Compound 14:2-(4-((3-(allyloxy)-3-oxopropyl)(3-(tert-butoxy)-3-oxopropyl)amino)-4-oxobutanamido)ethanesulfonicAcid

A mixture of compound 13 (1.3 g, 3.64 mmol) in THF (12 mL) and DSC (1.14g, 4.36 mmol) and DIEA (1.84 mL, 10.91 mmol) was stirred for 5 h at RT.After this time, a solution of taurine (718.75 mg, 5.46 mmol) in water(9 mL) was added and the medium was stirred at RT overnight. At thattime, DSC (1.14 g, 4.36 mmol) and DIEA (1.84 mL, 10.91 mmol) were addedand the medium was stirred at RT overnight. At this time, taurine (2.4g, 18.2 mmol) in water (5 mL) were added and the reaction medium wasstirred for 1 h at RT then concentrated and purified by flashchromatography on 70 g of C18-grafted silica gel (gradient elutionCH₃CN/H₂O) to give 1.28 g of compound 14 (76%).

RMN ¹H (400 MHz, δ in ppm, DMSO-d6): 50/50 conformer mixture; 1.39 (s,4.5H); 1.40 (s, 4.5H); 2.26 (t, J=7.2 Hz, 2H); 2.38 (t, J=7.5 Hz, 1H);2.47 to 2.55 (partially masked m, 6H); 2.69 (t, J=7.5 Hz, 1H); 3.28 (m,2H); 3.40 (t, J=7.5 Hz, 1H); 3.44 (t, J=7.5 Hz, 1H); 3.52 (t, J=7.5 Hz,1H); 3.58 (t, J=7.5 Hz, 1H); 4.53 (m, 1H); 4.58 (m, 1H); 5.19 to 5.35(m, 2H); 5.91 (m, 1H); 7.70 (t large, J=6.5 Hz, 1H).

Compound 15:3-(N-(3-(allyloxy)-3-oxopropyl)-4-oxo-4-((2-sulfoethyl)-amino)butanamido)propanoicAcid

A solution of compound 14 (300 mg, 645.81 μmol) in DCM (5 mL) and TFA(484.57 μL, 6.46 mmol) was stirred at RT overnight. At this time, TFA(250 μL) was added. The reaction medium was stirred several hours andconcentrated in vacuo, then diluted with toluene and concentrated invacuo twice to give 260 mg of compound 15 (99%).

RMN ¹H (400 MHz, δ in ppm, DMSO-d6): 50/50 conformer mixture; 2.27 (t,J=7.2 Hz, 2H); 2.40 (t, J=7.6 Hz, 1H); 2.48 to 2.56 (partially masked m,6H); 2.68 (t, J=7.6 Hz, 1H); 3.28 (m, 2H); 3.40 (t, J=7.6 Hz, 1H); 3.46(t, J=7.6 Hz, 1H); 3.50 (t, J=7.6 Hz, 1H); 3.59 (t, J=7.6 Hz, 1H); 4.54(m, 1H); 4.57 (m, 1H); 5.18 to 5.35 (m, 2H); 5.91 (m, 1H); 7.65 (broadt, J=6.5 Hz, 1H). LCMS (D): ES m/z=409 [M+H]⁺; t_(R)=1.49 min.

Compound 16:(4S,7S)-12-(3-(allyloxy)-3-oxopropyl)-1-(4-((2R,3R)-3-((S)-1-((3S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13-en-16-yl)ethyl)oxiran-2-yl)phenyl)-7-isopropyl-4-methyl-3,6,9,13,16-pentaoxo-2,5,8,12,17-pentaazanonadecane-19-sulfonicAcid

Under argon, to a solution of compound 15 (28.22 mg, 69.09 μmol) in DMF(3 mL) were added DIEA (23.30 μL, 138.18 μmol), HOBt (9.72 mg, 69.09μmol), EDC (12.49 μL, 69.09 μmol) and a solution of compound 11 (60 mg,69.09 μmol) in DMF (2 mL). The reaction medium was stirred at RTovernight. To complete the reaction, compound 15 (28.22 mg, 69.09 μmol),DIEA (23.30 μL, 138.18 μmol), HOBt (9.72 mg, 69.09 μmol), EDC (12.49 μL,69.09 μmol) were added and the reaction medium was stirred at RT for 1d. At this time, the reaction medium was concentrated in vacuo andpurified by flash chromatography on 70 g of C18-grafted silica gel(gradient elution CH₃CN/H₂O) to give 24 mg of compound 16 (24%).

RMN ¹H (500 MHz, δ in ppm, DMSO-d6): 60/40 conformer mixture; 0.79 (d,J=7.0 Hz, 6H); 0.80 to 0.87 (m, 6H); 1.00 (s, 3H); 1.04 (d, J=7.0 Hz,3H); 1.11 (s, 3H); 1.23 (d, J=7.0 Hz, 3H); 1.30 (m, 1H); 1.52 to 1.63(m, 2H); 1.80 (m, 1H); 1.95 (m, 1H); 2.27 (m, 3H); 2.34 to 2.58(partially masked m, 6H); 2.63 to 2.73 (m, 2H); 2.93 to 3.03 (m, 3H);3.25 to 3.59 (partially masked m, 9H); 3.81 (s, 3H); 3.88 (d, J=1.4 Hz,1H); 4.10 to 4.33 (m, 5H); 4.53 (m, 1.2H); 4.56 (m, 0.8H); 4.91 (m, 1H);5.10 (m, 1H); 5.20 (m, 1H); 5.29 (qd, J=1.8 and 17.3 Hz, 0.6H); 5.31(qd, J=1.8 and 17.3 Hz, 0.4H); 5.79 (dd, J=1.8 and 15.4 Hz, 1H); 5.90(m, 1H); 6.47 (ddd, J=3.8, 11.8 and 15.4 Hz, 1H); 7.05 (d, J=8.7 Hz,1H); 7.18 (dd, J=2.4 and 8.7 Hz, 1H); 7.24 (m, 5H); 7.29 (d, J=2.4 Hz,1H); 7.69 (t, J=5.8 Hz, 1H); 7.97 (d, J=8.8 Hz, 0.4H); 8.04 (d, J=7.7Hz, 0.4H); 8.07 (d, J=8.8 Hz, 0.6H); 8.11 (d, J=7.7 Hz, 0.6H); 8.31 (t,J=6.3 Hz, 1H); 8.38 (d, J=8.3 Hz, 1H); 11.83 (broad m, 1H). LCMS (D): ESm/z=1256 [M−H]⁻; t_(R)=2.58 min.

Example 4:(4S,7S)-1-(4-((2R,3R)-3-((S)-1-((3S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13-en-16-yl)ethyl)oxiran-2-yl)phenyl)-7-isopropyl-4-methyl-3,6,9-trioxo-12-(4-oxo-4-((2-sulfoethyl)amino)butanoyl)-2,5,8,12-tetraazapentadecan-15-oicAcid

Under argon, to a solution of compound 16 (24 mg, 19.06 μmol) in CH₃CN(2 mL) were added DMF (0.5 mL), 1,3-dimethylbarbituric acid (9.11 mg,57.19 μmol) and tetrakis(triphenylphosphine)palladium(0) (1.11 mg, 0.953μmol). The reaction medium was stirred for 1 h at RT, then concentratedin vacuo and purified by flash chromatography on 10 g of C18-graftedsilica gel (gradient elution CH₃CN/H₂O) to give 16 mg of example 4(70%).

RMN ¹H (500 MHz, δ in ppm, DMSO-d6): 60/40 conformer mixture; 0.79 (d,J=7.0 Hz, 6H); 0.80 to 0.86 (m, 6H); 0.99 (s, 3H); 1.04 (d, J=7.0 Hz,3H); 1.12 (s, 3H); 1.24 (d, J=7.0 Hz, 3H); 1.31 (m, 1H); 1.52 to 1.62(m, 2H); 1.80 (m, 1H); 1.95 (m, 1H); 2.21 to 2.31 (m, 3H); 2.34 to 2.59(partially masked m, 8H); 2.62 to 2.75 (m, 2H); 2.94 to 3.05 (m, 3H);3.25 to 3.58 (partially masked m, 7H); 3.81 (s, 3H); 3.88 (d, J=1.9 Hz,1H); 4.09 to 4.33 (m, 5H); 4.90 (m, 1H); 5.10 (m, 1H); 5.79 (d, J=15.5Hz, 1H); 6.47 (ddd, J=4.0, 11.4 and 15.5 Hz, 1H); 7.05 (d, J=8.7 Hz,1H); 7.17 (dd, J=2.2 and 8.7 Hz, 1H); 7.21 to 7.26 (m, 5H); 7.29 (d,J=2.2 Hz, 1H); 7.69 (m, 1H); 7.97 (d, J=9.0 Hz, 0.4H); 8.04 (d, J=7.7Hz, 0.4H); 8.08 (d, J=9.0 Hz, 0.6H); 8.12 (d, J=7.7 Hz, 0.6H); 8.31 (t,J=6.5 Hz, 1H); 8.38 (d, J=8.2 Hz, 1H); 12.2 (broad m, 1H). LCMS (A): ESm/z=609.5 [M+2H]²⁺; m/z=1216 [M−H]⁻; m/z=1218 [M+H]⁺; t_(R)=1.72 min.

Example 5:(4S,7S)-1-(4-((2R,3R)-3-((S)-1-((3S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13-en-16-yl)ethyl)oxiran-2-yl)phenyl)-12-(3-((2,5-dioxopyrrolidin-1-yl)oxy)-3-oxopropyl)-7-isopropyl-4-methyl-3,6,9,13,16-pentaoxo-2,5,8,12,17-pentaazanonadecane-19-sulfonicAcid

Under argon, a mixture of example 4 (16 mg, 13.13 μmol) in DMF (1 mL),DSC (4.12 mg, 15.75 μmol) and DIEA (2.66 μL, 15.75 μmol) was stirred atRT overnight.

At this time, the reaction medium was concentrated in vacuo and purifiedby flash chromatography on 5 g of C18-grafted silica gel (gradientelution CH₃CN/H₂O) to give 12.5 mg of example 5 (73%).

RMN ¹H (500 MHz, δ in ppm, DMSO-d6): 60/40 conformer mixture; 0.79 (d,J=7.0 Hz, 6H); 0.80 to 0.86 (m, 6H); 0.99 (s, 3H); 1.04 (d, J=7.0 Hz,3H); 1.11 (s, 3H); 1.23 (d, J=7.0 Hz, 3H); 1.31 (m, 1H); 1.51 to 1.62(m, 2H); 1.80 (m, 1H); 1.93 (m, 1H); 2.21 to 2.31 (m, 3H); 2.35 to 2.59(partially masked m, 6H); 2.62 to 2.74 (m, 2H); 2.81 (broad s, 4H); 2.85to 3.05 (m, 4H); 3.20 to 3.68 (partially masked m, 8H); 3.81 (s, 3H);3.88 (d, J=1.9 Hz, 1H); 4.10 to 4.33 (m, 5H); 4.90 (m, 1H); 5.10 (m,1H); 5.79 (d, J=15.5 Hz, 1H); 6.46 (ddd, J=4.0, 11.4 and 15.5 Hz, 1H);7.05 (d, J=8.7 Hz, 1H); 7.18 (dd, J=2.2 and 8.7 Hz, 1H); 7.21 to 7.26(m, 5H); 7.29 (d, J=2.2 Hz, 1H); 7.69 (m, 1H); 7.97 (d, J=9.0 Hz, 0.4H);8.05 (d, J=7.7 Hz, 0.4H); 8.08 (d, J=9.0 Hz, 0.6H); 8.12 (d, J=7.7 Hz,0.6H); 8.31 (t, J=6.5 Hz, 1H); 8.38 (d, J=8.2 Hz, 1H). LCMS (A): ESm/z=658 [M+2H]²⁺; m/z=1313 [M−H]⁻; m/z=1315 [M+H]⁺; t_(R)=1.82 min.

Example 6: hu2H11_R35-74-Ex5

The general method described previously was used for the preparation ofexample 6. 15 mg of hu2H11_R35-74 were reacted with 42 μL of a 10 mMsolution of example 5 in DMA (4 eq.) for 1 h 20 then were successivelyadded 42 μL of a 10 mM solution of example 5 in DMA (4 eq.) for 1 h 20,53 μL of a 10 mM solution of example 5 in DMA (5 eq.) for 1 h 30 andfinally 42 μL of a 10 mM solution of example 5 in DMA (4 eq.) for 1 h.After purification on Superdex 200 pg in buffer B pH 6.5+20% NMP,concentration on Amicon Ultra-15, buffer exchange on PD-10 in buffer BpH 6.5+5% NMP and filtration on 0.22 μm PVDF filter, 3.33 mg of example6 were obtained as a colorless limpid solution at a concentration of1.33 mg/mL with a DAR of 4.05 (HRMS), a monomeric purity of 99% and aglobal yield of 22%.

SEC-HRMS: m/z=150568 (D1); m/z=151768 (D2); m/z=152971 (D3); m/z=154172(D4); m/z=155375 (D5); m/z=156574 (D6); m/z=157944 (D7); m/z=158984(D8).

Synthesis of Example 7: sulfo-Val-Ala-aza-crypto Benzylic Amine

Compound 17: tert-Butyl L-valyl-L-alaninate

To a solution of Fmoc-Val-OH (CAS number [68858-20-8], 3 g, 8.66 mmol)in DCM (60 mL) were added, under Ar, EDC (2.02 g, 12.99 mmol), HOBt(1.46 g, 10.40 mmol) and L-alanine tert-butyl ester hydrochloride (CASnumber [13404-22-3], 1.61 g, 8.66 mmol). The reaction medium was stirredfor 1 d at RT, quenched with H₂O (30 mL) and extracted with DCM (3×30mL). The combined organic phases were washed with brine, dried overMgSO₄, filtered, concentrated in vacuo to give 5 g of Fmoc-protecteddipeptide as a white solid (quant.). To a solution of this intermediate(5 g, 8.66 mmol) in DCM (50 mL) was added piperidine (8.64 mL, 86.63mmol). The reaction medium was stirred for 1 h at RT and concentrated invacuo. The crude product was diluted with Et₂O and filtered to give 1.77g of a solid that was further purified by two consecutive flashchromatographies on 120 g and 50 g of silica gel (gradient elutionDCM/MeOH) to give 500 mg of compound 17 as a white solid (24%).

RMN ¹H (400 MHz, δ in ppm, DMSO-d6): 0.79 (d, J=6.9 Hz, 3H); 0.89 (d,J=6.9 Hz, 3H); 1.24 (d, J=7.3 Hz, 3H); 1.40 (s, 9H); 1.62 (broad s, 2H);1.86 (m, 1H); 2.95 (d, J=5.2 Hz, 1H); 4.15 (m, 1H); 8.09 (broad d, J=7.3Hz, 1H).

Compound 18:(5S,8S)-13-(3-(allyloxy)-3-oxopropyl)-8-isopropyl-2,2,5-trimethyl-4,7,10,14,17-pentaoxo-3-oxa-6,9,13,18-tetraazaicosane-20-sulfonicAcid

To a solution of compound 15 (250 mg, 612.1 μmol) in DMF (6 mL) wereadded, under Ar, DIEA (309.7 μL, 1.84 mmol) and DSC (163.34 mg, 612.1μmol). The reaction medium was stirred for 1 h at RT then was added asolution of compound 17 (149.56 mg, 612.1 μL) in DMF (1 mL). Thereaction medium was stirred for 2 h at RT and purified by reverse phasechromatography on a 5 μm C18 column 30×100 mm (gradient elutionMeCN+0.07% TFA/H₂O+0.07% TFA) to give 215 mg of compound 18 as a whitefoam (55%).

RMN ¹H (400 MHz, δ in ppm, DMSO-d6): 60/40 conformer mixture; 0.84 (d,J=6.9 Hz 3H); 0.87 (d, J=6.9 Hz, 3H); 1.12 (d, J=7.3 Hz, 3H); 1.39 (s,9H); 1.92 (m, 1H); 2.20 to 2.70 (partially masked m, 10H); 3.28 (m, 2H);3.34 to 3.60 (m, 4H); 4.10 (m, 1H); 4.20 (m, 1H); 4.53 (td, J=1.6 and5.5 Hz, 1.2H); 4.57 (td, J=1.6 and 5.5 Hz, 0.8H); 5.20 (m, 1H); 5.30 (m,1H); 5.90 (m, 1H); 7.69 (broad t, J=6.4 Hz, 1H); 7.91 (d, J=9.6 Hz,0.4H); 8.04 (d, J=9.6 Hz, 0.6H); 8.23 (d, J=7.2 Hz, 0.4H); 8.30 (d,J=7.2 Hz, 0.6H).

Compound 19:(3-(N-(3-(allyloxy)-3-oxopropyl)-4-oxo-4-((2-sulfoethyl)amino)-butanamido)-propanoyl)-L-valyl-L-alanine

To a solution of compound 18 (215 mg, 338.72 μmol) in DCM (5 mL) wasadded TFA (508.3 μL, 6.77 mmol). The reaction medium was stirred for 1 dat RT, concentrated in vacuo and co-evaporated with toluene (3×) to give200 mg of compound 19 as a white foam (quant.).

Compound 20:(4S,7S)-12-(3-(allyloxy)-3-oxopropyl)-1-(4-((2R,3R)-3-((S)-1-((3S,7S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-6,6,7-trimethyl-3-neopentyl-2,5,9,12-tetraoxo-1-oxa-4,8,11-triazacyclohexadec-13-en-16-yl)ethyl)oxiran-2-yl)phenyl)-7-isopropyl-4-methyl-3,6,9,13,16-pentaoxo-2,5,8,12,17-pentaazanona-decane-19-sulfonicAcid

To a solution of compound 19 (200 mg, 345.6 μmol) in DMF (9 mL), wereadded DSC (108 mg, 413.2 μmol) and DIEA (200 μL, 1.185 mmol). Thereaction medium was stirred for 6 h at RT then were added added DSC (108mg, 413.2 μmol) and DIEA (200 μL, 1.185 mmol). The reaction medium wasstirred at RT overnight then were added DSC (50 mg, 191.3 μmol) and DIEA(100 μL, 592.5 μmol) and stirring carried on for 1 h at RT. To 3 mL ofthe solution of activated ester were added(3S,7S,10R,16S,E)-16-((S)-1-((2R,3R)-3-(4-(aminomethyl)phenyl)oxiran-2-yl)ethyl)-10-(3-chloro-4-methoxybenzyl)-6,6,7-trimethyl-3-neopentyl-1-oxa-4,8,11-triazacyclo-hexadec-13-ene-2,5,9,12-tetraone(that can be synthesized as described in PCT/EP2016/076603 starting frommethyl (3S)-3-amino-2,2-dimethylbutanoate, [MFCD09256689], 70 mg, 96.51μmol), EDC (17 μL, 77.8 μmol) and HOBt (19 mg, 124 μmol). The reactionmedium was stirred for 1 h at RT, concentrated in vacuo and purified byflash chromatography on 10 g of C18-modified silica gel (gradientelution MeCN/H₂O) to give 30 mg of compound 20 as a white lacquer (24%).

RMN ¹H (600 MHz, δ in ppm, DMSO-d6): 0.79 to 0.90 (m, 15H); 0.98 (m,3H); 1.00 (s, 3H); 1.03 (d, J=7.3 Hz, 3H); 1.19 (s, 3H); 1.24 (split d,J=7.0 Hz, 3H); 1.34 (d, J=14.1 Hz, 1H); 1.81 (m, 1H); 1.91 (m, 1H); 1.98(dd, J=9.3 and 14.1 Hz, 1H); 2.25 (m, 3H); 2.38 (m, 1H); 2.50 (partiallymasked m, 5H); 2.60 (m, 1H); 2.68 (m, 1H); 2.91 (m, 1H); 2.96 (m, 2H);3.25 to 3.58 (partially masked m, 9H); 3.80 (s, 3H); 3.91 (m, 1H); 4.00to 4.35 (m, 6H); 4.53 (m, 2H); 5.03 (m, 1H); 5.20 (m, 1H); 5.30 (m, 1H);5.89 (d, J=16.1 Hz, 1H); 5.91 (m, 1H); 6.44 (ddd, J=5.0, 10.5 and 16.1Hz, 1H); 7.03 (d, J=8.7 Hz, 1H); 7.19 to 7.28 (m, 5H); 7.31 (s, 1H);7.69 (m, 1H); 7.86 (d, J=8.2 Hz, 1H); 7.91 (m, 1H); 7.95 to 8.17 (m,2H); 8.32 (m, 1H); 8.39 (m, 1H). LCMS (A): ES m/z=1283 [M−H]⁻; m/z=1285[M+H]⁺; m/z=1307 [M+Na]⁺; t_(R)=1.93 min.

Example 7:(4S,7S)-1-(4-((2R,3R)-3-((S)-1-((3S,7S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-6,6,7-trimethyl-3-neopentyl-2,5,9,12-tetraoxo-1-oxa-4,8,11-triazacyclohexadec-13-en-16-yl)ethyl)-oxiran-2-yl)phenyl)-7-isopropyl-4-methyl-3,6,9-trioxo-12-(4-oxo-4-((2-sulfoethyl)amino)butanoyl)-2,5,8,12-tetraazapentadecan-15-oicAcid

To a solution of compound 20 (30 mg, 23.33 μmol) in DCM (2 mL) and DMF(0.3 mL) were added, under Ar, tetrakis(triphenylphosphine)palladium(0)(1.38 mg, 1.17 μmol) and 1,3-dimethyl-barbituric acid (11.15 mg, 69.99μmol). The reaction medium was stirred for 1 h at RT and purified byreverse phase chromatography on 1 g of C18-modified silica gel (gradientelution MeCN/H₂O) to give 15 mg of a white powder that was furtherpurified on silica gel (gradient elution DCM/MeOH/H₂O) to give 7 mg ofexample 7 as a white lacquer (24%).

Synthesis of Examples 8 & 9: PEG4-Val-Ala-PABA-C52 Benzylic Amine andNHS Ester of PEG4-Val-Ala-PABA-C52 Benzylic Amine

Compound 21: (9H-fluoren-9-yl)methyl((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)-amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate

To a solution of Fmoc-Val-Ala-OH (CAS number [150114-97-9], 1 g, 2.44mmol) in DCM (30 mL) and MeOH (15 mL) were added 4-aminobenzyl alcohol(612 mg, 4.87 mmol) and EEDQ (1.22 g, 4.87 mmol). The reaction mediumwas stirred at RT for 24 h then additional 4-aminobenzyl alcohol (612mg, 4.87 mmol) and EEDQ (1.22 g, 4.87 mmol) were added and the stirringcarried on 2 h. The reaction medium was then concentrated in vacuo; Et₂Owas added to the crude product, the mixture was stirred and filtered togive a brown solid that was diluted in Et₂O and the mixture was stirredat RT overnight. It was then filtered and the precipitate was dried togive 1.3 g of compound 21 as a brown solid (quant.).

RMN ¹H (400 MHz, δ in ppm, DMSO-d6): 0.86 (d, J=7.0 Hz, 3H); 0.89 (d,J=7.0 Hz, 3H); 1.30 (d, J=7.0 Hz, 3H); 2.00 (m, 1H); 3.91 (m, 1H); 4.19to 4.34 (m, 4H); 4.43 (d, J=5.7 Hz, 2H); 5.08 (t, J=5.7 Hz, 1H); 7.23(d, J=7.9 Hz, 2H); 7.31 (t, J=7.9 Hz, 2H); 7.40 (m, 3H); 7.53 (d, J=9.1Hz, 2H); 7.73 (t, J=8.6 Hz, 2H); 7.89 (d, J=7.9 Hz, 2H); 8.16 (d, J=7.3Hz, 1H); 9.91 (s, 1H). LCMS (A): ES m/z=292; m/z=393; m/z=516 [M+H]⁺;t_(R)=1.21 min.

Compound 22: (9H-fluoren-9-yl)methyl((S)-3-methyl-1-(((S)-1-((4-((((4-nitrophenoxy)carbonyl)-oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxobutan-2-yl)carbamate

To a solution of compound 21 (1.3 g, 2.52 mmol) in DMF (10 mL), wasadded, under Ar, DIEA (2.55 mL, 15.13 mmol), the solution was cooleddown at 0° C. before the addition of 4-nitrophenyl chloroformate (1.57g, 7.56 mmol). The reaction medium was stirred for 2 h at RT. 20 mL ofH₂O were added and the medium was extracted twice with EtOAc (10 mL).The combined organic phases were washed with brine, dried over MgSO₄,filtered, concentrated in vacuo and purified by flash chromatography on120 g of silica gel (gradient elution DCM/MeOH) to give 600 mg ofcompound 22 as a solid (35%).

RMN ¹H (400 MHz, δ in ppm, DMSO-d6): 0.86 (d, J=7.0 Hz, 3H); 0.89 (d,J=7.0 Hz, 3H); 1.31 (d, J=7.0 Hz, 3H); 1.99 (m, 1H); 3.91 (m, 1H); 4.18to 4.35 (m, 3H); 4.43 (m, 1H); 5.24 (s, 2H); 7.31 (t, J=7.9 Hz, 2H);7.35 to 7.44 (m, 5H); 7.56 (d, J=9.1 Hz, 2H); 7.63 (d, J=8.6 Hz, 2H);7.74 (m, 2H); 7.88 (d, J=7.9 Hz, 2H); 8.19 (d, J=7.3 Hz, 1H); 8.31 (d,J=9.1 Hz, 2H); 10.07 (s, 1H).

Compound 23: 4-((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)benzyl4-((2R,3R)-3-((S)-1-((3S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13-en-16-yl)ethyl)oxiran-2-yl)benzylcarbamate

To a solution of(3S,10R,16S,E)-16-((S)-1-((2R,3R)-3-(4-(aminomethyl)phenyl)oxiran-2-yl)ethyl)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-1,4-dioxa-8,11-diazacyclohexadec-13-ene-2,5,9,12-tetraone(the synthesis of which was described in WO2011001052, compound 77, 100mg, 143.2 μmol) in THF (5 mL), were added, under argon compound 22 (117mg, 171.9 μmol) and DIEA (60.4 μL, 358 μmol). The reaction medium wasstirred at RT under Ar overnight. Then piperidine (142.9 μL, 1.43 mmol)was added and the reaction medium stirred for 1 h at RT. Afterconcentrating in vacuo, the crude medium was purified by flashchromatography on 5 g of silica gel (gradient elution DCM/MeOH) to give100 mg of compound 23 as a white solid (73%).

Compound 24: allyl15-(3-(((S)-1-(((S)-1-((4-((((4-((2R,3R)-3-((S)-1-((3S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclo-hexadec-13-en-16-yl)ethyl)oxiran-2-yl)benzyl)carbamoyl)oxy)-methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-oxopropyl)-14-oxo-2,5,8,11-tetraoxa-15-azaoctadecan-18-oate

To a solution of compound 23 (100 mg, 98.3 μmol) in THF (5 mL), wereadded, under Ar, compound 9 (50.8 mg, 98.3 μmol) and DIEA (24.9 μL,147.4 μmol). The reaction medium was stirred for 1 h at RT, concentratedin vacuo and purified by flash chromatography on 5 g of silica gel(gradient elution DCM/MeOH) to give 128 mg of compound 24 as a whitesolid (91%).

RMN ¹H (500 MHz, δ in ppm, DMSO-d6): 60:40 conformer mixture; 0.78 (d,J=6.8 Hz, 6H); 0.82 (d, J=7.0 Hz, 1.8H); 0.84 (d, J=7.0 Hz, 1.2H); 0.86(d, J=7.0 Hz, 1.8H); 0.88 (d, J=7.0 Hz, 1.2H); 1.00 (s, 3H); 1.04 (d,J=7.0 Hz, 3H); 1.11 (s, 3H); 1.30 (m, 4H); 1.57 (m, 2H); 1.80 (m, 1H);1.95 (m, 1H); 2.26 (m, 1H); 2.40 (m, 2H); 2.52 to 2.72 (m, 6H); 2.95 to3.04 (m, 3H); 3.22 (s, 3H); 3.30 (m, 1H); 3.38 to 3.63 (m, 18H); 3.80(s, 3H); 3.87 (d, J=2.3 Hz, 1H); 4.15 to 4.28 (m, 4H); 4.40 (m, 1H);4.51 to 4.57 (m, 2H); 4.90 (dd, J=3.8 and 9.8 Hz, 1H); 4.98 (s, 2H);5.10 (m, 1H); 5.20 (m, 1H); 5.30 (m, 1H); 5.80 (d, J=16.0 Hz, 1H); 5.91(m, 1H); 6.47 (ddd, J=4.7, 10.5 and 16.0 Hz, 1H); 7.05 (d, J=8.6 Hz,1H); 7.16 (dd, J=2.5 and 8.6 Hz, 1H); 7.29 to 7.32 (m, 8H); 7.58 (m,2H); 7.76 (t, J=6.5 Hz, 1H); 7.92 (d, J=9.0 Hz, 0.4H); 8.04 (d, J=9.0Hz, 0.6H); 8.15 (d, J=7.0 Hz, 0.4H); 8.21 (d, J=7.0 Hz, 0.6H); 8.32 (d,J=8.0 Hz, 1H); 9.88 (s, 0.4H); 9.93 (s, 0.6H). LCMS (A): ES m/z=677;m/z=1418 [M+H]⁺; m/z=1462 [M−H+HCO₂H]⁻; t_(R)=1.21 min.

Example 8:15-(3-(((S)-1-(((S)-1-((4-((((4-((2R,3R)-3-((S)-1-((3S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13-en-16-yl)ethyl)oxiran-2-yl)benzyl)carbamoyl)oxy)methyl)-phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-oxopropyl)-14-oxo-2,5,8,11-tetraoxa-15-azaoctadecan-18-oicAcid

To a solution of compound 24 (58 mg, 40.9 μmol) in DCM (20 mL), wereadded, under Ar, 1,3-dimethylpyrimidine-2,4,6(1H, 3H, 5H)-trione (19.5mg, 122.6 μmol) and tetrakis (triphenylphosphine)palladium (0) (2.4 mg,2.0 μmol). The reaction medium was stirred for 3 h at RT, concentratedin vacuo and purified by flash chromatography on 5 g of silica gel(gradient elution DCM/MeOH) to give 53 mg of example 8 as a white solid(94%).

RMN ¹H (500 MHz, δ in ppm, DMSO-d6): conformer mixture; 0.78 (d, J=6.8Hz, 6H); 0.86 (m, 6H); 0.99 (s, 3H); 1.04 (d, J=7.0 Hz, 3H); 1.11 (s,3H); 1.30 (m, 1H); 1.38 (m, 3H); 1.57 (m, 2H); 1.79 (m, 1H); 2.04 (m,1H); 2.10 to 2.59 (partially masked m, 7H); 2.69 (m, 2H); 2.93 to 3.03(m, 3H); 3.22 (s, 3H); 3.30 (masked m, 1H); 3.40 to 3.64 (m, 18H); 3.80(s, 3H); 3.87 (d, J=2.3 Hz, 1H); 4.05 to 4.27 (m, 4H); 4.36 (m, 1H);4.90 (dd, J=3.8 and 9.8 Hz, 1H); 4.97 (s, 2H); 5.10 (m, 1H); 5.80 (d,J=16.0 Hz, 1H); 5.90 (ddd, J=4.7, 10.5 and 16.0 Hz, 1H); 6.46 (ddd,J=4.7, 10.5 and 16.0 Hz, 1H); 7.05 (d, J=8.6 Hz, 1H); 7.16 (dd, J=2.5and 8.6 Hz, 1H); 7.20 to 7.32 (m, 8H); 7.68 (m, 2H); 7.82 (m, 1H); 8.40to 8.65 (m, 2H); 9.21 (m, 1H); 10.20 (s, 1H). LCMS (A): ES m/z=637;m/z=1376 [M−H]⁻; m/z=1378 [M+H]⁺; t_(R)=1.3 min.

Example 9: 2,5-dioxopyrrolidin-1-yl15-(3-(((S)-1-(((S)-1-((4-((((4-((2R,3R)-3-((S)-1-((3S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13-en-16-yl)ethyl)oxiran-2-yl)benzyl)carbamoyl)oxy)methyl)-phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-oxopropyl)-14-oxo-2,5,8,11-tetraoxa-15-azaoctadecan-18-oate

To a solution of example 8 (49 mg, 35.5 μmol) in DMF (2 mL), were added,under Ar, DSC (13.9 mg, 53.3 μmol) and DIEA (9.0 μL, 53.3 μmol). Thereaction medium was stirred at RT overnight. Additional DSC (13.9 mg,53.3 μmol) and DIEA (9.0 μL, 53.3 μmol) were added and the stirringcarried over 8 h. The reaction medium was concentrated in vacuo andpurified by flash chromatography on 10 g of diol-modified silica gel(gradient elution DCM/iPrOH) to give 20 mg of example 9 as a white solid(38%).

RMN ¹H (500 MHz, δ in ppm, DMSO-d6): 70:30 conformer mixture; 0.78 (d,J=6.8 Hz, 6H); 0.82 (d, J=7.0 Hz, 2.1H); 0.84 (d, J=7.0 Hz, 0.9H); 0.86(d, J=7.0 Hz, 2.1H); 0.88 (d, J=7.0 Hz, 0.9H); 1.00 (s, 3H); 1.05 (d,J=7.0 Hz, 3H); 1.11 (s, 3H); 1.27 (m, 1H); 1.30 (m, 3H); 1.57 (m, 2H);1.80 (m, 1H); 1.95 (m, 1H); 2.25 (m, 1H); 2.40 (m, 1H); 2.52 to 2.75 (m,7H); 2.80 (s, 4H); 2.87 to 3.08 (m, 3H); 3.22 (s, 3H); 3.32 (masked m,1H); 3.40 to 3.68 (m, 18H); 3.80 (s, 3H); 3.88 (d, J=2.3 Hz, 1H); 4.16to 4.28 (m, 4H); 4.38 (m, 1H); 4.90 (dd, J=3.8 and 9.8 Hz, 1H); 4.97 (s,2H); 5.10 (m, 1H); 5.79 (d, J=16.0 Hz, 1H); 6.46 (ddd, J=4.7, 10.5 and16.0 Hz, 1H); 7.05 (d, J=8.6 Hz, 1H); 7.16 (dd, J=2.5 and 8.6 Hz, 1H);7.20 to 7.31 (m, 8H); 7.58 (m, 2H); 7.78 (t, J=6.0 Hz, 1H); 7.94 (d,J=9.0 Hz, 0.3H); 8.08 (d, J=9.0 Hz, 0.7H); 8.19 (d, J=7.0 Hz, 0.3H);8.25 (d, J=7.0 Hz, 0.7H); 8.35 (d, J=8.0 Hz, 1H); 9.90 (s, 0.3H); 9.95(s, 0.7H). LCMS (A): ES m/z=698; m/z=1475 [M+H]⁺; t_(R)=1.35 min.

Synthesis of Example 10: PEG7-Val-Ala-PABA-C52 Benzylic Amine

Compound 25: tert-butyl 2,5,8,11,14,17,20-heptaoxatricosan-23-oate

To a solution of hexaethylene glycol monomethyl ether (926.0 μL, 3.24mmol) in THF (3 mL), was added, under Ar, Na (0.7 mg, 32.4 μmol). Thereaction medium was stirred for 1 h at RT until complete dissolution ofNa then tert-butyl acrylate (570 μL, 3.89 mmol) was added and thestirring carried on at RT overnight. The reaction medium was thenconcentrated in vacuo, diluted with H₂O and extracted with EtOAc (3×).The combined organic phases were washed with brine, dried over MgSO₄,filtered and concentrated in vacuo to give 1.04 g of compound 25 as acolorless oil (76%).

RMN ¹H (400 MHz, 8 in ppm, DMSO-d6): 1.40 (s, 9H); 2.40 (t, J=6.4 Hz,2H); 3.23 (s, 3H); 3.43 (m, 2H); 3.47 to 3.54 (m, 22H); 3.59 (t, J=6.4Hz, 2H).

Compound 26: 2,5,8,11,14,17,20-heptaoxatricosan-23-oic Acid

To a solution of compound 25 (1.04 g, 2.45 mmol) in DCM (15 mL) wasadded TFA (1.84 mL, 24.5 mmol). The reaction medium was stirred at RTovernight, concentrated in vacuo, diluted with DCM and co-evaporatedwith toluene (3×) to give 1 g of compound 26 as a colorless oil(quant.).

Compound 27: 2,5,8,11,14,17,20-heptaoxatricosan-23-oyl Chloride

To compound 26 (400 mg, 1.09 mmol) was added MsCI (1.27 mL, 16.3 mmol);the reaction medium was heated 3 h at 60° C., concentrated in vacuo,diluted with DCM and concentrated in vacuo (3×) to give 420 mg ofcompound 27 as a colorless oil (quant.).

Compound 28: allyl24-(3-(tert-butoxy)-3-oxopropyl)-23-oxo-2,5,8,11,14,17,20-heptaoxa-24-azaheptacosan-27-oate

To a solution of compound 3 (200 mg, 777.2 μmol) in DCM (5 mL) was addedDIEA (262 μL, 1.55 mmol). The reaction medium was cooled down at 0° C.and a solution of compound 27 (360.8 mg, 932.7 μmol) in DCM (2 mL) wasadded dropwise at 0° C. The reaction medium was stirred for 1 h at RTand concentrated in vacuo. The crude product was purified by twoconsecutive flash chromatographies on 12 g and 10 g of silica gel(gradient elution DCM/MeOH) to give 72 mg of compound 28 (15%).

RMN ¹H (400 MHz, δ in ppm, DMSO-d6): 50:50 conformer mixture; 1.39 (s,4.5H); 1.40 (s, 4.5H); 2.22 to 2.75 (partially masked m, 6H); 3.23 (s,3H); 3.38 to 3.63 (m, 30H); 4.55 (m, 2H); 5.19 to 5.36 (m, 2H); 5.90 (m,1H).

Compound 29:24-(3-(allyloxy)-3-oxopropyl)-23-oxo-2,5,8,11,14,17,20-heptaoxa-24-azahepta-cosan-27-oicAcid

To a solution of compound 28 (72 mg, 118.5 μmol) in DCM (3 mL) was addedTFA (100 μL, 1.33 mmol); the reaction medium was stirred at RT overnightthen additional TFA (100 μL, 1.33 mmol) was added and the stirringcarried on for 1 d. The reaction medium was concentrated in vacuo,diluted with DCM and co-evaporated with toluene (3×) to give 66 mg ofcompound 29 (quant.).

Compound 30: allyl24-(3-((2,5-dioxopyrrolidin-1-yl)oxy)-3-oxopropyl)-23-oxo-2,5,8,11,14,17,20-heptaoxa-24-azaheptacosan-27-oate

To a solution of compound 29 (66 mg, 119.7 μmol) in THF (2 mL), wereadded, under Ar, DIEA (20.2 μL, 119.9 μmol) and DSC (34.4 mg, 131.6μmol); the reaction medium was stirred for 4 d at RT, concentrated invacuo and purified by flash chromatography on 900 mg of diol-modifiedsilica gel (gradient elution DCM/iPrOH) to give 35 mg of compound 30(45%).

Compound 31: allyl24-(3-(((S)-1-(((S)-1-((4-((((4-((2R,3R)-3-((S)-1-((3S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclo-hexadec-13-en-16-yl)ethyl)oxiran-2-yl)benzyl)carbamoyl)oxy)-methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-oxopropyl)-23-oxo-2,5,8,11,14,17,20-heptaoxa-24-azaheptacosan-27-oate

To a solution of compound 23 (45 mg, 44.2 μmol) in THF (5 mL) were addedcompound 30 (28.7 mg, 44.2 μmol) and DIEA (11.2 μL, 66.3 μmol). Thereaction medium was stirred at RT overnight, diluted with H₂O (10 mL)and extracted with EtOAc (3×10 mL). The combined organic phases werewashed with brine, dried over MgSO₄, filtered, concentrated in vacuo andpurified by flash chromatography on 5 g of silica gel (gradient elutionDCM/MeOH) to give 35 mg of compound 31 as a white solid (51%).

RMN ¹H (500 MHz, δ in ppm, DMSO-d6): 60:40 conformer mixture; 0.80(split d, J=6.8 Hz, 6H); 0.82 (d, J=7.0 Hz, 1.8H); 0.84 (d, J=7.0 Hz,1.2H); 0.86 (d, J=7.0 Hz, 1.8H); 0.88 (d, J=7.0 Hz, 1.2H); 1.00 (s, 3H);1.04 (d, J=7.0 Hz, 3H); 1.11 (s, 3H); 1.28 (m, 1H); 1.30 (m, 3H); 1.56(m, 2H); 1.79 (m, 1H); 1.95 (m, 1H); 2.27 (m, 1H); 2.35 to 2.60(partially masked m, 6H); 2.62 to 2.73 (m, 2H); 2.93 to 3.05 (m, 3H);3.23 (s, 3H); 3.33 (masked m, 1H); 3.38 to 3.64 (m, 30H); 3.80 (s, 3H);3.87 (d, J=2.3 Hz, 1H); 4.15 to 4.27 (m, 4H); 4.38 (m, 1H); 4.52 to 4.58(m, 2H); 4.90 (dd, J=3.8 and 9.8 Hz, 1H); 4.97 (s, 2H); 5.10 (m, 1H);5.50 (m, 1H); 5.30 (m, 1H); 5.80 (d, J=16.0 Hz, 1H); 5.90 (m, 1H); 6.47(ddd, J=4.7, 10.5 and 16.0 Hz, 1H); 7.05 (d, J=8.6 Hz, 1H); 7.15 (dd,J=2.5 and 8.6 Hz, 1H); 7.20 to 7.32 (m, 8H); 7.59 (m, 2H); 7.78 (t,J=6.5 Hz, 1H); 7.95 (d, J=9.0 Hz, 0.4H); 8.07 (d, J=9.0 Hz, 0.6H); 8.18(d, J=7.0 Hz, 0.4H); 8.27 (d, J=7.0 Hz, 0.6H); 8.36 (d, J=8.0 Hz, 1H);9.90 (s, 0.4H); 9.95 (s, 0.6H). LCMS (A): ES m/z=698; m/z=1552 [M+H]⁺;m/z=1595 [M−H+HCO₂H]⁻; t_(R)=1.39 min.

Example 10:24-(3-(((S)-1-(((S)-1-((4-((((4-((2R,3R)-3-((S)-1-((3S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13-en-16-yl)ethyl)oxiran-2-yl)benzyl)carbamoyl)oxy)methyl)-phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-oxopropyl)-23-oxo-2,5,8,11,14,17,20-heptaoxa-24-azaheptacosan-27-oicAcid

To a solution of compound 31 (30 mg, 19.3 μmol) in DCM (5 mL) wereadded, under Ar, dimethylbarbituric acid (9.24 mg, 58.0 μmol) andtetrakis(triphenylphosphine)palladium (0) (1.13 mg, 0.97 μmol). Thereaction medium was stirred for 3 h at RT then concentrated in vacuo andpurified by two consecutive flash chromatographies on 5 g and 0.9 g ofsilica gel (gradient elution DCM/MeOH) to give 14 mg of example 10 as awhite solid (50%).

RMN ¹H (500 MHz, δ in ppm, DMSO-d6): 50:50 conformer mixture; 0.78(split d, J=6.8 Hz, 6H); 0.81 to 0.89 (m, 6H); 1.00 (s, 3H); 1.04 (d,J=7.0 Hz, 3H); 1.12 (s, 3H); 1.28 (m, 1H); 1.30 (d, J=7.0 Hz, 1.5H);1.32 (d, J=7.0 Hz, 1.5H); 1.51 to 1.62 (m, 2H); 1.79 (m, 1H); 1.96 (m,1H); 2.26 (m, 1H); 2.33 to 2.60 (partially masked m, 6H); 2.61 to 2.73(m, 2H); 2.93 to 3.03 (m, 3H); 3.22 (s, 3H); 3.30 (masked m, 1H); 3.38to 3.62 (m, 30H); 3.80 (s, 3H); 3.87 (d, J=2.3 Hz, 1H); 4.12 to 4.29 (m,4H); 4.39 (m, 1H); 4.90 (dd, J=3.8 and 9.8 Hz, 1H); 4.96 (s, 2H); 5.10(m, 1H); 5.78 (d, J=16.0 Hz, 1H); 6.46 (ddd, J=4.7, 10.5 and 16.0 Hz,1H); 7.05 (d, J=8.6 Hz, 1H); 7.16 (dd, J=2.5 and 8.6 Hz, 1H); 7.20 to7.32 (m, 8H); 7.59 (m, 2H); 7.79 (m, 1H); 8.04 (m, 0.5H); 8.10 (d, J=9.0Hz, 0.5H); 8.25 to 8.41 (m, 2H); 9.95 (s, 0.5H); 9.98 (s, 0.5H); 12.20(m, 1H). LCMS (D): ES m/z=769; m/z=1511 [M+H]⁺; tR=3.16 min.

Synthesis of Examples 11 & 12: PEG24-Val-Ala-PABA-C52 Benzylic Amine andNHS Ester of PEG24-Val-Ala-PABA-C52 Benzylic Amine

Compound 32: allyl75-(3-(tert-butoxy)-3-oxopropyl)-74-oxo-2,5,8,11,14,17,20,23,26,29,32,35,38,41,47,50,53,56,59,62,65,68,71-tetracosaoxa-75-azaoctaheptacontan-78-oate

To a solution of compound 3 (45 mg, 174.9 μmol) in THF (5 mL) wereadded, under Ar, MEO-DPEG(24)-NHS (212.4 mg, 174.9 μmol) and DIEA (44.2μL, 262.3 μmol).

The reaction medium was stirred at RT overnight then concentrated invacuo and purified by flash chromatography on 20 g of silica gel(gradient elution DMC/MeOH) to give 125 mg of a mixture of compound 32(60%) and MEO-DPEG(24)-NHS (40%). This mixture was diluted in THF (3mL), 15 mg of compound 3 and 20 μL of DIEA were added; the reactionmedium was stirred at RT overnight then concentrated in vacuo andpurified by flash chromatography on 12 g of silica gel to give 105 mg ofcompound 32 (44%).

RMN ¹H (400 MHz, δ in ppm, DMSO-d6): 1.40 (s, 9H); 2.25 to 2.78 (m, 6H);3.22 (s, 3H); 3.32 to 3.70 (m, 98H); 4.52 (m, 2H); 5.20 (m, 1H); 5.30(m, 1H); 5.90 (m, 1H). LCMS (A): ES m/z=679 [M+2H]²⁺; m/z=1356 [M+H]⁺;m/z=1400 [M−H+HCO₂H]⁻; t_(R)=1.15 min.

Compound 33:75-(3-(allyloxy)-3-oxopropyl)-74-oxo-2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71-tetracosaoxa-75-azaoctaheptacontan-78-oicAcid

To a solution of compound 32 (105 mg, 77.4 μmol) in DCM (2 mL) was addedTFA (116.2 μL, 1.55 mmol); the reaction medium was stirred at RTovernight, 150 μL of TFA were then added and the reaction carried on onemore day. The reaction medium was then concentrated in vacuo, dilutedwith DCM and co-evaporated with toluene (3×) to give 110 mg of crudeproduct that were purified by RP-HPLC on a 5 μm C18 SunFire column(Waters, 30×100 mm, gradient elution MeCN+0.07% TFA/H₂O+0.07% TFA) toprovide 60 mg of compound 33 (60%).

RMN ¹H (400 MHz, δ in ppm, DMSO-d6): 50:50 conformer mixture; 2.37 to2.60 (partially masked m, 6H); 3.23 (s, 3H); 3.32 to 3.44 (partiallymasked m, 38H); 3.47 to 3.52 (m, 58H); 3.60 (m, 2H); 4.54 (m, 2H); 5.22(dm, J=10.5 Hz, 1H); 5.30 (dm, J=17.3 Hz, 1H); 5.84 to 5.98 (m, 1H);12.32 (broad m, 1H).

Compound 34: allyl75-(3-(((S)-1-(((S)-1-((4-((((4-((2R,3R)-3-((S)-1-((3S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclo-hexadec-13-en-16-yl)ethyl)oxiran-2-yl)benzyl)carbamoyl)oxy)-methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-oxopropyl)-74-oxo-2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71-tetracosaoxa-75-azaoctaheptacontan-78-oate

To a solution of compound 33 (30 mg, 23.1 μmol) in THF (5 mL) wereadded, under Ar, DIEA (11.7 μL, 69.2 μmol) and DSC (6.03 mg, 23.1 μmol).The reaction medium was stirred for 3 h at RT then was added compound 23(23.5 mg, 23.1 μmol) and the stirring carried on 1 h. The reactionmedium was concentrated in vacuo and purified by flash chromatography on5 g of silica gel to give 23 mg of compound 34 (40%).

RMN ¹H (500 MHz, δ in ppm, DMSO-d6): 60:40 conformer mixture: 0.80(split d, J=6.8 Hz, 6H); 0.82 (d, J=7.0 Hz, 1.8H); 0.84 (d, J=7.0 Hz,1.2H); 0.86 (d, J=7.0 Hz, 1.8H); 0.88 (d, J=7.0 Hz, 1.2H); 1.00 (s, 3H);1.04 (d, J=7.0 Hz, 3H); 1.11 (s, 3H); 1.28 (m, 1H); 1.30 (d, J=7.0 Hz,3H); 1.51 to 1.62 (m, 2H); 1.80 (m, 1H); 1.96 (m, 1H); 2.26 (m, 1H);2.38 (m, 1H); 2.45 to 2.61 (partially masked m, 5H); 2.61 to 2.72 (m,2H); 2.93 to 3.05 (m, 3H); 3.23 (s, 3H); 3.32 (masked m, 1H); 3.40 to3.77 (m, 98H); 3.80 (s. 3H); 3.88 (d, J=2.3 Hz, 1H; 4.14 to 4.27 (m,4H); 4.39 (m, 1H); 4.51 to 4.60 (m, 2H); 4.90 (dd, J=3.8 and 9.8 Hz,1H); 4.97 (s, 2H); 5.10 (m, 1H); 5.18 to 5.33 (m, 2H); 5.78 (d, J=16.0Hz, 1H); 5.90 (m, 1H); 6.45 (m, 1H); 7.05 (d, J=8.6 Hz, 1H); 7.16 (dd,J=2.5 and 8.6 Hz, 1H); 7.20 to 7.32 (m, 8H); 7.59 (m, 2H); 7.77 (t,J=6.5 Hz, 1H); 7.95 (d, J=9.0 Hz, 0.4H); 8.07 (d, J=9.0 Hz, 0.6H); 8.19(d, J=7.0 Hz, 0.4H); 8.25 (d, J=7.0 Hz, 0.6H); 8.36 (d, J=8.0 Hz, 1H);9.90 (s, 0.4H); 9.96 (s, 0.6H).

Example 11:75-(3-(((S)-1-(((S)-1-((4-((((4-((2R,3R)-3-((S)-1-((3S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13-en-16-yl)ethyl)oxiran-2-yl)benzyl)carbamoyl)oxy)methyl)-phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-oxopropyl)-74-oxo-2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71-tetracosaoxa-75-azaoctaheptacontan-78-oic Acid

To a solution of compound 34 (37 mg, 161 μmol) in DCM were added, underAr, 1,3-dimethylbarbituric acid (5.13 mg, 32.2 μmol) andtetrakis(triphenylphosphine)palladium(0) (0.94 mg, 0.80 μmol); thereaction medium was stirred at RT for 2 h then concentrated in vacuo andpurified by flash chromatography on 5 g of diol-modified silica gel(gradient elution DCM/MeOH) to give 16 mg of example 11 (44%).

RMN ¹H (500 MHz, δ in ppm, DMSO-d6): 0.77 (d, J=7.0 Hz, 6H); 0.80 to0.90 (m, 6H); 0.99 (s, 3H); 1.04 (d, J=7.0 Hz, 3H); 1.11 (s, 3H); 1.28(m, 1H); 1.30 (d, J=7.0 Hz, 1.5H); 1.32 (d, J=7.0 Hz, 1.5 H); 1.52 to1.61 (m, 2H); 1.79 (m, 1H); 1.98 (m, 1H); 2.22 to 2.58 (partially maskedm, 7H); 2.68 (m, 2H); 2.92 to 3.04 (m, 3H); 3.21 (s, 3H); 3.30(partially masked m, 1H); 3.37 to 3.67 (m, 98H); 3.81 (s, 3H); 3.88 (s,1H); 4.08 to 4.28 (m, 4H); 4.39 (m, 1H); 4.90 (m, 1H); 4.98 (s, 2H);5.10 (m, 1H); 5.80 (d, J=16.0 Hz, 1H); 6.48 (ddd, J=4.7, 10.5 and 16.0Hz, 1H); 7.04 (d, J=8.6 Hz, 1H); 7.16 (broad d, J=8.6 Hz, 1H); 7.20 to7.32 (m, 8H); 7.57 to 7.67 (m, 2H); 7.80 (m, 1H); 8.20 (m, 1H); 8.35 to8.70 (m, 2H); 10.08 (m, 1H); 12.30 (broad m, 1H). LCMS (D): ES m/z=698;m/z=2259 [M+H]⁺; t_(R)=3.14 min.

Example 12: 2,5-dioxopyrrolidin-1-yl75-(3-(((S)-1-(((S)-1-((4-((((4-((2R,3R)-3-((S)-1-((3S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13-en-16-yl)ethyl)oxiran-2-yl)benzyl)-carbamoyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-oxopropyl)-74-oxo-2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71-tetracosaoxa-75-azaoctaheptacontan-78-oate

To a solution of example 11 (16 mg, 7.1 μmol) in DCM were added DIEA(1.2 μL, 7.1 μmol) and DSC (1.8 mg, 7.1 μmol). The reaction medium wasstirred for 3 h at RT then concentrated in vacuo and purified by flashchromatography on 1.3 g of diol-modified silica gel (gradient elutionDCM/iPrOH) to give 5 mg of a mixture of example 12 (30%) and example 11(70%).

LCMS (D): ES m/z=698; m/z=2356 [M+H]⁺; t_(R)=3.2 min.

Synthesis of Example 13: sulfo-PEG4-Val-Ala-PABA-C52 Benzylic Amine

Compound 35:4-((3-(allyloxy)-3-oxopropyl)(3-(tert-butoxy)-3-oxopropyl)amino)-4-oxobutanoicAcid

To a solution of compound 3 (100 mg, 388.6 μmol) in DCM (5 mL) was addedsuccinic anhydride (78.6 mg, 777.2 μmol). The reaction medium wasstirred at RT overnight then concentrated in vacuo and purified by flashchromatography on 4 g of silica gel (gradient elution DCM/MeOH) to give102 mg of compound 35 as a colorless oil (73%).

Compound 36:1-ammonio-15-oxo-3,6,9,12-tetraoxa-16-azaoctadecane-18-sulfonate

To a solution of 15-(Boc-amino)-4,7,10,13-tetraoxapentadecanoic acid(500 mg, 1.3 mmol) in THF (5 mL) were added, under Ar, DIEA (438.4 μL,2.6 mmol) and DSC (381.6 mg, 1.43 mmol). The reaction medium was stirredfor 6 h at RT then 50 mg of DSC were added and the stirring was carriedon at RT overnight. At that time, taurine (821.6 mg, 6.5 mmol) and H₂O(1 mL) were added to the reaction medium. The reaction medium wasstirred at RT overnight, concentrated in vacuo and purified bypreparative LCMS on a 5 μm C18 SunFire column (Waters, 30×100 mm,gradient elution MeCN+0.07% TFA/H₂O+0.07% TFA) to give 341 mg ofcompound 36 (70%).

RMN ¹H (400 MHz, δ in ppm, DMSO-d6): 2.28 (t, J=6.5 Hz, 2H); 2.60 (m,2H); 2.99 (m, 2H); 3.32 (m, 2H); 3.45 to 3.65 (m, 16H); 7.77 (broad m,4H). IR spectrum as a KBr pellet; main absorption bands in reciprocalcentimeters: 3000; 1780; 1648; 1556; 1200-1170; 1200; 1100; 1040.

Compound 37:24-(3-(tert-butoxy)-3-oxopropyl)-4,20,23,27-tetraoxo-7,10,13,16,28-pentaoxa-3,19,24-triazahentriacont-30-ene-1-sulfonate

To a solution of compound 35 (100 mg, 279.8 μmol) in THF were added DIEA(141.6 μL, 839.4 μmol) and DSC (73.1 mg, 279.8 μmol). The reactionmixture was stirred for 2 h at RT then a solution of compound 36 (104.2mg, 279.8 μmol) in DMF and DIEA (141.6 μL, 839.4 μmol) were added. Thereaction medium was stirred for 2 h at RT, concentrated in vacuo andpurified by preparative LCMS on a 5 μm C18 SunFire column (Waters,30×100 mm, gradient elution MeCN+0.07% TFA/H₂O+0.07% TFA) to give 118 mgof compound 37 (60%).

RMN ¹H (400 MHz, δ in ppm, DMSO-d6): 50:50 conformer mixture; 1.39 (s,4.5H); 1.40 (s, 4.5H); 2.27 (t, J=6.5 Hz, 2H); 2.31 (t, J=7.0 Hz, 2H);2.38 (t, J=7.0 Hz, 1H); 2.45 to 2.56 (masked m, 6H); 2.68 (m, 1H); 3.18(q, J=6.0 Hz, 2H); 3.29 (m, 2H); 3.36 to 3.60 (m, 20H); 4.51 to 4.59 (m,2H); 5.17 to 5.25 (m, 2H); 5.85 to 5.95 (m, 1H); 7.75 (t, J=6.0 Hz, 1H);7.88 (t, J=6.0 Hz, 1H).

Compound 38:24-(2-carboxyethyl)-4,20,23,27-tetraoxo-7,10,13,16,28-pentaoxa-3,19,24-triazahentriacont-30-ene-1-sulfonate

To a solution of compound 37 (118 mg, 166.0 μmol) in DCM (5 mL) wasadded TFA (249 μL, 3.32 mmol). The reaction medium was stirred at RTovernight, concentrated in vacuo, diluted with DCM and co-evaporatedwith toluene (3×) to give 100 mg of compound 38 as a colorless oil(92%).

Compound 39:(2S,5S)-10-(3-(allyloxy)-3-oxopropyl)-1-((4-((((4-((2R,3R)-3-((S)-1-((3S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13-en-16-yl)ethyl)oxiran-2-yl)benzyl)carbamoyl)oxy)methyl)phenyl)amino)-5-isopropyl-2-methyl-1,4,7,11,14,30-hexaoxo-18,21,24,27-tetraoxa-3,6,10,15,31-pentaazatritria-contane-33-sulfonicAcid

To a solution of compound 38 (100 mg, 152.7 μmol) in THF (3 mL) and DMF(1 mL) were added DIEA (77 μL, 458.2 μmol) and DSC (39.9 mg, 152.7μmol). The reaction medium was stirred for 2 h at RT before the additionof a solution of compound 23 (124 mg, 121.9 μmol) in THF (1 mL) and DMF(0.5 mL) and DIEA (100 μL). The reaction medium was stirred for 2 h atRT, concentrated in vacuo and purified by reverse phase flashchromatography on 75 g of C18-modified silica gel (gradient elutionH₂O/MeCN) to give 60 mg of compound 39 as a white solid (24%).

RMN ¹H (500 MHz, δ in ppm, DMSO-d6): 60:40 conformer mixture; 0.78 (d,J=6.8 Hz, 6H); 0.82 (d, J=7.0 Hz, 1.8H); 0.84 (d, J=7.0 Hz, 1.2H); 0.86(d, J=7.0 Hz, 1.8H); 0.88 (d, J=7.0 Hz, 1.2H); 0.99 (s, 3H); 1.04 (d,J=7.0 Hz, 3H); 1.11 (s, 3H); 1.28 (m, 1H); 1.30 (d, J=7.0 Hz, 3H); 1.50to 1.62 (m, 2H); 1.80 (m, 1H); 1.96 (m, 1H); 2.20 to 2.55 (partiallymasked m, 11H); 2.61 to 2.75 (m, 2H); 2.94 to 3.05 (m, 3H); 3.10 to 3.65(partially masked, 27H); 3.80 (s, 3H); 3.87 (d, J=2.3 Hz, 1H); 4.13 to4.28 (m, 4H); 4.38 (m, 1H); 4.53 (m, 1.2H); 4.56 (m, 0.8H); 4.90 (dd,J=3.8 and 9.8 Hz, 1H); 4.97 (s, 2H); 5.10 (m, 1H); 5.18 to 5.33 (m, 2H);5.80 (d, J=16.0 Hz, 1H); 5.90 (m, 1H); 6.47 (ddd, J=4.7, 10.5 and 16.0Hz, 1H); 7.05 (d, J=8.6 Hz, 1H); 7.16 (dd, J=2.5 and 8.6 Hz, 1H); 7.20to 7.32 (m, 8H); 7.59 (m, 2H); 7.72 (t, J=6.5 Hz, 1H); 7.79 (t, J=6.0Hz, 1H); 7.89 (t, J=6.5 Hz, 1H); 7.95 (d, J=9.0 Hz, 0.4H); 8.08 (d,J=9.0 Hz, 0.6H); 8.19 (d, J=7.0 Hz, 0.4H); 8.25 (d, J=7.0 Hz, 0.6H);8.36 (d, J=8.0 Hz, 1H); 9.90 (s, 0.4H); 9.95 (s, 0.6H). LCMS (D): ESm/z=913; m/z=1654 [M+H]⁺; t_(R)=3.87 min.

Example 13:24-(3-(((S)-1-(((S)-1-((4-((((4-((2R,3R)-3-((S)-1-((3S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13-en-16-yl)ethyl)oxiran-2-yl)benzyl)carbamoyl)oxy)methyl)-phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-oxopropyl)-4,20,23-trioxo-1-sulfo-7,10,13,16-tetraoxa-3,19,24-triazaheptacosan-27-oicAcid

To a solution of compound 39 (60 mg, 36.3 μmol) in DMF (5 mL) were added1,3-dimethylbarbituric acid (17.3 mg, 108.8 μmol) and tetrakis(triphenylphosphine)palladium(0) (2.1 mg, 1.8 μmol). The reaction mediumwas stirred at RT overnight, concentrated in vacuo and purified by twoflash chromatographies on 30 g and 20 g of C18-modified silica gel togive two batches of 6 mg of example 13 as a white solid (20% globalyield).

RMN ¹H (500 MHz, δ in ppm, DMSO-d6): 60:40 conformer mixture; 0.78 (d,J=6.8 Hz, 6H); 0.81 to 0.89 (m, 6H); 1.00 (s, 3H); 1.04 (d, J=7.0 Hz,3H); 1.11 (s, 3H); 1.28 (m, 1H); 1.30 (d, J=7.0 Hz, 3H); 1.58 (m, 2H);1.80 (m, 1H); 1.96 (m, 1H); 2.21 to 2.58 (partially masked m, 11H); 2.69(m, 2H); 2.93 to 3.68 (partially masked m, 28H); 3.80 (s, 3H); 3.88 (d,J=2.3 Hz, 1H); 4.11 to 4.28 (m, 4H); 4.38 (m, 1H); 4.90 (dd, J=3.8 and9.8 Hz, 1H); 4.98 (s, 2H); 5.10 (m, 1H); 5.79 (d, J=16.0 Hz, 1H); 6.47(ddd, J=4.7, 10.5 and 16.0 Hz, 1H); 7.05 (d, J=8.6 Hz, 1H); 7.17 (dd,J=2.5 and 8.6 Hz, 1H); 7.20 to 7.32 (m, 8H); 7.59 (m, 2H); 7.72 (t,J=6.5 Hz, 1H); 7.79 (t, J=6.0 Hz, 1H); 7.89 (t, J=6.5 Hz, 1H); 7.97 (d,J=9.0 Hz, 0.4H); 8.08 (d, J=9.0 Hz, 0.6H); 8.19 (d, J=7.0 Hz, 0.4H);8.25 (d, J=7.0 Hz, 0.6H); 8.36 (d, J=8.0 Hz, 1H); 9.90 (s, 0.4H); 9.94(s, 0.6H); 12.15 (broad m, 1H). LCMS (A): ES m/z=807.5 [M+2H]²⁺;m/z=1612 [M−H]⁻; m/z=1614 [M+H]⁺; t_(R)=1.72 min.

Synthesis of Examples 14 to 16: glutaryl-Val-PEG4Gln-C52 Benzylic Amine,NHS Ester of glutaryl-Val-PEG4Gln-C52 Benzylic Amine and CorrespondingADC

Compound 40:(S)-4-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-5-(tert-butoxy)-5-oxopentanoicAcid

To a solution of H-Glu-OtBu (CAS number [45120-30-7], 959 mg, 4.7 mmol)and NaHCO₃ (400 mg, 4.77 mmol) in H₂O (80 mL), was added dropwise, undermagnetic stirring, a solution of (S)-2,5-dioxopyrrolidin-1-yl2-((((9H-fluoren-9-yl)methoxy)-carbonyl)amino)-3-methylbutanoate (2 g,4.6 mmol) in THF (80 mL). The reaction medium was stirred at RTovernight. The medium was concentrated in vacuo, then saturated aqueousNaHCO₃ (1 L) was added, followed by extraction with Et₂O (2×250 mL),organic phases were excluded. The aqueous phase was acidified with QS ofaqueous 1N HCl to reach pH 2, and extracted with DCM (3×250 mL), thecombined organic phases were dried over MgSO₄, filtered and concentratedin vacuo to give 2.05 g compound 40 (85%).

RMN ¹H (400 MHz, δ in ppm, DMSO-d6): 0.87 (d, J=6.9 Hz, 3H); 0.90 (d,J=6.9 Hz, 3H); 1.38 (s, 9H); 1.73 to 2.02 (m, 3H); 2.24 to 2.30 (m, 2H);3.90 (dd, J=7.3 and 9.0 Hz, 1H); 4.14 (m, 1H); 4.19 to 4.32 (m, 3H);7.27 to 7.45 (m, 5H); 7.75 (t, J=7.6 Hz, 2H); 7.89 (d, J=7.6 Hz, 2H);8.22 (broad d, J=7.0 Hz, 1H); 12.05 (broad s, 1H).

Compound 41: (S)-tert-butyl18-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-15-oxo-2,5,8,11-tetraoxa-14-azanonadecan-19-oate

To a solution of compound 40 (200 mg, 381 μmol) in DCM (6 mL) wereadded, under magnetic stirring, PEG4-NH₂ (CAS number [85030-56-4], 115mg, 554.9 μmol), HOBt (82 mg, 606.8 μmol) and EDC (100 μL, 565 μmol).The reaction medium was stirred at RT overnight. The crude medium waspurified twice by flash chromatography on silica gel (gradient elutionDCM/MeOH) to give 190 mg of compound 41 (70%).

RMN ¹H (400 MHz, δ in ppm, DMSO-d6): 0.87 (d, J=6.8 Hz, 3H); 0.90 (d,J=6.8 Hz, 3H); 1.38 (s, 9H); 1.72 to 1.93 (m, 2H); 1.99 (m, 1H); 2.14(t, J=7.8 Hz, 2H); 3.18 (m, 2H); 3.23 (s, 3H); 3.37 (t, J=6.0 Hz, 2H);3.41 (m, 2H); 3.46 to 3.53 (m, 10H); 3.92 (dd, J=7.1 and 8.8 Hz, 1H);4.09 (m, 1H); 4.17 to 4.34 (m, 3H); 7.32 (m, 3H); 7.41 (t, J=7.6 Hz,2H); 7.70 to 7.81 (m, 3H); 7.88 (d, J=7.6 Hz, 2H); 8.18 (d, J=7.3 Hz,1H). LCMS (A): ES m/z=714 [M+H]⁺; m/z=758 [M−H+HCO₂H]⁻; t_(R)=1.41 min.

Compound 42:(S)-18-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-15-oxo-2,5,8,11-tetraoxa-14-azanonadecan-19-oicAcid

To compound 41 (190 mg, 266 μmol) was added, under magnetic stirring, a4M solution of HCl in 1,4-dioxane (5 mL). The reaction medium wasstirred at RT for 4 h. At this time, the reaction medium wasconcentrated in vacuo and purified by flash chromatography on 15 g ofsilica gel (gradient elution DCM/MeOH/NH₄OH) to give 70 mg of compound42 (40%).

RMN ¹H (400 MHz, δ in ppm, DMSO-d6): 0.84 (d, J=6.6 Hz, 3H); 0.86 (d,J=6.6 Hz, 3H); 1.70 to 1.90 (m, 2H); 2.01 to 2.15 (m, 3H); 3.15 (m, 2H);3.23 (s, 3H); 3.36 (t, J=6.4 Hz, 2H); 3.42 (m, 2H); 3.45 to 3.53 (m,10H); 3.80 (broad m, 1H); 3.83 (dd, J=6.8 and 8.7 Hz, 1H); 4.19 to 4.34(m, 3H); 7.33 (m, 2H); 7.41 (t, J=7.6 Hz, 2H); 7.59 (broad m, 2H); 7.73(d, J=7.6 Hz, 1H); 7.76 (d, J=7.6 Hz, 1H); 7.89 (d, J=7.6 Hz, 2H); 8.00(broad m, 1H). LCMS (A): ES m/z=656 [M−H]⁻; m/z=658 [M+H]⁺; RT=1.19 min.

Compound 43:(S)-2-((S)-2-amino-3-methylbutanamido)-N1-(4-((2R,3R)-3-((S)-1-((3S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13-en-16-yl)ethyl)oxiran-2-yl)benzyl)-N5-(2,5,8,11-tetraoxatridecan-13-yl)pentanediamide

To a solution of(E)-(3S,10R,16S)-16-{(S)-1-[(2R,3R)-3-(4-aminomethyl-phenyl)-oxiranyl]-ethyl}-10-(3-chloro-4-methoxy-benzyl)-3-isobutyl-6,6-dimethyl-1,4-dioxa-8,11-diaza-cyclohexadec-13-ene-2,5,9,12-tetraone (thesynthesis of which was described in WO2011001052, compound 77, 58 mg, 83μmol) in DMF (5 mL), under magnetic stirring, were added compound 42 (70mg, 106 μmol), HOBt (20 mg, 148 μmol) and EDC (23 μL, 130 μmol). Thereaction medium was stirred at RT for 24 h. At this time, piperidine (80μL, 810 μmol) was added to the medium, and stirring was maintained for 1h. Then, the reaction medium was concentrated in vacuo and purified byflash chromatography on 20 g silica gel (gradient elution DCM/MeOH) togive 60 mg of compound 43 (65%).

RMN ¹H (500 MHz, δ in ppm, DMSO-d6): 0.77 (d, J=6.9 Hz, 3H); 0.79 (d,J=6.3 Hz, 6H); 0.87 (d, J=6.9 Hz, 3H); 1.00 (s, 3H); 1.04 (d, J=6.4 Hz,3H); 1.12 (s, 3H); 1.31 (m, 1H); 1.52 to 1.63 (m, 2H); 1.72 to 1.98 (m,4H); 2.05 to 2.15 (m, 2H); 2.26 (m, 1H); 2.62 to 2.73 (m, 2H); 2.94 to3.04 (m, 4H); 3.18 (q, J=5.9 Hz, 2H); 3.23 (s, 3H); 3.33 (partiallymasked m, 1H); 3.39 (t, J=5.9 Hz, 2H); 3.42 (m, 2H); 3.47 to 3.52 (m,10H); 3.81 (s, 3H); 3.87 (d, J=1.4 Hz, 1H); 4.21 to 4.36 (m, 4H); 4.91(dd, J=3.6 and 9.6 Hz, 1H); 5.10 (dd, J=5.5 and 11.8 Hz, 1H); 5.79 (d,J=15.3 Hz, 1H); 6.46 (ddd, J=3.8, 11.6 and 15.3 Hz, 1H); 7.05 (d, J=8.5Hz, 1H); 7.17 (dd, J=1.9 and 8.5 Hz, 1H); 7.21 to 7.25 (m, 5H); 7.28 (d,J=1.9 Hz, 1H); 7.89 (t, J=5.5 Hz, 1H); 8.07 (m large, 1H); 8.37 (d,J=8.0 Hz, 1H); 8.45 (t, J=5.9 Hz, 1H). LCMS (A): ES m/z=558 [M+2H]²⁺;m/z=1115 [M+H]⁺; m/z=1159 [M−H+HCO₂H]⁻; t_(R)=0.96 min.

Example 14:(18S,21S)-18-((4-((2R,3R)-3-((S)-1-((3S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13-en-16-yl)ethyl)oxiran-2-yl)benzyl)carbamoyl)-21-isopropyl-15,20,23-trioxo-2,5,8,1I-tetraoxa-14,19,22-triazaheptacosan-27-oic Acid

To a solution of compound 43 (55 mg, 49.3 μmol) in DMF (3 mL) was addedglutaric anhydride (9 mg, 79 μmol), the reaction medium was stirred atRT for 2 h. At this time, the medium was concentrated in vacuo andpurified by flash chromatography on 10 g of silica gel (gradient elutionDCM/MeOH) to give 38 mg of example 14 (62%).

RMN ¹H (500 MHz, δ in ppm, DMSO-d6): 0.81 (split d, J=6.6 Hz, 6H); 0.85(d, J=6.3 Hz, 3H); 0.87 (d, J=6.3 Hz, 3H); 0.98 (s, 3H); 1.03 (d, J=6.6Hz, 3H); 1.11 (s, 3H); 1.35 (m, 1H); 1.52 to 2.34 (m, 15H); 2.71 (m,2H); 2.89 to 3.02 (m, 3H); 3.09 to 3.21 (m, 2H); 3.23 (s, 3H); 3.30 to3.40 (partially masked m, 3H); 3.42 (m, 2H); 3.46 to 3.51 (m, 10H); 3.81(s, 3H); 3.87 (s, 1H); 4.03 to 4.24 (m, 4H); 4.37 (m, 1H); 4.96 (m, 1H);5.11 (m, 1H); 5.81 (d, J=15.3 Hz, 1H); 6.46 (ddd, J=3.3, 11.4 and 15.3Hz, 1H); 7.04 (d, J=8.5 Hz, 1H); 7.19 (dd, J=1.9 and 8.5 Hz, 1H); 7.22(d, J=8.3 Hz, 2H); 7.27 (d, J=8.3 Hz, 2H); 7.30 (d, J=1.9 Hz, 1H); 7.32(m, 1H); 7.85 to 8.99 (broad m, 5H); 12.12 (broad m, 1H). LCMS (A): ESm/z=615 [M+2H]²⁺; m/z=1227 [M−H]⁻; m/z=1229 [M+H]⁺; t_(R)=1.26 min.

Example 15: (18S,21 S)-2,5-dioxopyrrolidin-1-yl18-((4-((2R,3R)-3-((S)-1-((3S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13-en-16-yl)ethyl)oxiran-2-yl)benzyl)carbamoyl)-21-isopropyl-15,20,23-trioxo-2,5,8,1I-tetraoxa-14,19,22-triazaheptacosan-27-oate

To a solution of example 14 (32 mg, 26 μmol) in DMF (3 mL), undermagnetic stirring, were added DSC (8 mg, 31.2 μmol) and DIEA (8.6 μL, 52μmol). The reaction medium was stirred at RT overnight. Then, 4 mg ofDSC were added and stirring at RT was maintained for 1 h. At this time,the reaction medium was diluted with MeTHF (10 mL), washed with H₂O (5mL). The aqueous phase was extracted with MeTHF (3×5 mL). The combinedorganic phases were dried over MgSO₄, filtered and concentrated invacuo. The medium was purified by flash chromatography on 4 g of silicagel (gradient elution DCM/iPrOH) to give 28 mg of example 15 (81%).

RMN ¹H (500 MHz, δ in ppm, DMSO-d6): 0.78 (d, J=6.6 Hz, 6H); 0.83 (d,J=6.9 Hz, 3H); 0.85 (d, J=6.9 Hz, 3H); 1.00 (s, 3H); 1.04 (d, J=6.4 Hz,3H); 1.12 (s, 3H); 1.30 (m, 1H); 1.52 to 1.64 (m, 2H); 1.75 to 1.93 (m,5H); 1.98 (m, 1H); 2.12 (m, 2H); 2.22 to 2.34 (m, 3H); 2.62 to 2.72 (m,4H); 2.81 (s, 4H); 2.93 to 3.04 (m, 3H); 3.18 (q, J=6.0 Hz, 2H); 3.23(s, 3H); 3.33 (partially masked m, 1H); 3.39 (t, J=6.0 Hz, 2H); 3.42 (m,2H); 3.47 to 3.52 (m, 10H); 3.81 (s, 3H); 3.87 (d, J=1.6 Hz, 1H); 4.15(dd, J=6.6 and 8.2 Hz, 1H); 4.19 to 4.32 (m, 4H); 4.91 (dd, J=3.6 and9.6 Hz, 1H); 5.10 (m, 1H); 5.79 (d, J=15.3 Hz, 1H); 6.47 (ddd, J=3.7,11.3 and 15.3 Hz, 1H); 7.05 (d, J=8.5 Hz, 1H); 7.17 (dd, J=2.2 and 8.5Hz, 1H); 7.21 to 7.26 (m, 5H); 7.28 (d, J=2.2 Hz, 1H); 7.85 (t, J=6.0Hz, 1H); 7.92 (d, J=8.2 Hz, 1H); 8.08 (d, J=7.7 Hz, 1H); 8.32 (t, J=6.0Hz, 1H); 8.37 (d, J=8.0 Hz, 1H). LCMS (A): ES m/z=1326 [M+H]⁺; m/z=1348[M+Na]⁺; m/z=1370 [M−H+HCO₂H]⁻; t_(R)=1.31 min.

Example 16: hu2H11_R35-74-Ex15

The general method described previously was used for the preparation ofexample 16. 28.8 mg of hu2H11_R35-74 were reacted with 115.2 μL of a10.05 mM solution of example 15 in DMA (6 eq.) for 2 h 30. Afterpurification on Superdex 200 pg in buffer B pH 6.5+10% NMP,concentration on Amicon Ultra-15, dilution in buffer B pH 6.5 to a finalconcentration of NMP at 5% and filtration on 0.22 μm PVDF filter, 16.12mg of ADC were obtained that contained 2.2% of residual example 14. Thisbatch was concentrated on Amicon Ultra-15 and purified on Sephadex G25to provide 13.16 mg of example 16 as a colorless limpid solution at aconcentration of 1.4 mg/mL with a DAR of 2.8 (HRMS), a monomeric purityof 99.7% and a global yield of 43%.

SEC-HRMS: m/z=149398 (naked mAb); m/z=150610 (D1); m/z=151822 (D2);m/z=153035 (D3); m/z=154248 (D4); m/z=155458 (D5); m/z=156672 (D6);m/z=1578881 (D7).

Synthesis of Examples 17 to 19: glutaryl-Val-GlucoseGln-C52 BenzylicAmine, NHS Ester of glutaryl-Val-GlucoseGln-C52 Benzylic Amine andCorresponding ADC

Compound 44:(2S,3S,4S,5R,6R)-2-(iodomethyl)-6-methoxytetrahydro-2H-pyran-3,4,5-triol

To a solution of methyl R-D-glucopyranoside hemihydrate (CAS number[7000-27-3], 1 g, 4.67 mmol), imidazole (642 mg, 9.34 mmol) andtriphenylphosphine (2.47 g, 9.33 mmol) in THF (50 mL) stirred at reflux,was added dropwise a solution of iodine (2.37 g, 9.33 mmol) in THF (10mL). Reflux was maintained for 3 h. After cooling at 0° C., the reactionmedium was filtered, the filtrate was concentrated in vacuo. The mixturewas purified by flash chromatography on 150 g of silica gel (gradientDCM/MeOH) to give 1 g of compound 44 (63%).

Compound 45:(2R,3S,4S,5R,6R)-2-(azidomethyl)-6-methoxytetrahydro-2H-pyran-3,4,5-triol

To a solution of compound 44 (1 g, 3.29 mmol) in DMF (10 mL), undermagnetic stirring, was added NaN₃ (322 mg, 4.93 mmol). The reactionmedium was heated at 50° C. for 16 h. At this time, after cooling at RT,the medium was concentrated in vacuo and purified by flashchromatography on 80 g of silica gel (gradient elution DCM/MeOH) to give550 mg of compound 45 (76%).

Compound 46:(2R,3S,4S,5R,6R)-2-(aminomethyl)-6-methoxytetrahydro-2H-pyran-3,4,5-triolhydrochloride

A solution of compound 45 (550 mg, 2.51 mmol) and triphenylphosphine(1.33 g, 5.02 mmol) in 1,4-dioxane (12 mL) and MeOH (3 mL) was stirredat RT for 30 min. At this time, water (1.5 mL) was added to the reactionmedium and stirring at RT was maintained overnight. The crude medium wasconcentrated in vacuo, then diluted with DCM (10 mL) and aqueous 1N HCl(5 mL), the aqueous phase was extracted with DCM (2×10 mL). The aqueousphase was concentrated in vacuo, then diluted with MeOH and concentratedin vacuo to afford 560 mg of compound 46 (97%).

Compound 47: (S)-1-tert-butyl 5-(2,5-dioxopyrrolidin-1-yl)2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)pentanedioate

To a solution of compound 40 (2 g, 3.81 mmol) in THF (100 mL) were addedsuccessively, under magnetic stirring, DIEA (1.29 mL, 7.62 mmol), DSC(1.95 g, 7.62 mmol) and DMF (20 mL). The reaction medium was stirred atRT under Ar overnight. After concentration in vacuo, the mixture waspurified by flash chromatography on 150 g of silica gel (elutionDCM/EtOAc) to give 1.67 g of compound 47 (71%).

RMN ¹H (400 MHz, δ in ppm, DMSO-d6): 0.89 (d, J=6.8 Hz, 3H); 0.91 (d,J=6.8 Hz, 3H); 1.39 (s, 9H); 1.84 to 2.10 (m, 3H); 2.65 to 2.77 (m, 2H);2.80 (s, 4H); 3.88 (dd, J=7.2 and 8.7 Hz, 1H); 4.18 to 4.31 (m, 4H);7.32 (m, 2H); 7.41 (m, 3H); 7.73 (d, J=7.6 Hz, 1H); 7.75 (d, J=7.6 Hz,1H); 7.88 (d, J=7.6 Hz, 2H); 8.31 (broad d, J=7.1 Hz, 1H). LCMS (A): ESm/z=566; m/z=622 [M+H]⁺; m/z=666 [M−H+HCO₂H]⁻; t_(R)=1.49 min.

Compound 48: (S)-tert-butyl2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-5-oxo-5-((((2R,3S,4S,5R,6R)-3,4,5-trihydroxy-6-methoxytetrahydro-2H-pyran-2-yl)methyl)amino)pentanoate

To a solution of compound 47 (500 mg, 804 μmol) in DMF (10 mL) wereadded, under magnetic stirring, compound 46 (185 mg, 805 μmol) and DIEA(165 μL, 978 μmol). The reaction medium was stirred at RT for 4 h. Atthis time, 37 mg of compound 46 and 35 μL of DIEA were added to thereaction medium, stirring was maintained for 1 h. Then, the medium wasconcentrated in vacuo and purified by flash chromatography on 50 g ofsilica gel (gradient elution DCM/MeOH) to give 480 mg of compound 48(85%).

RMN ¹H (400 MHz, δ in ppm, DMSO-d6): 0.87 (d, J=6.7 Hz, 3H); 0.89 (d,J=6.7 Hz, 3H); 1.38 (s, 9H); 1.70 to 2.04 (m. 3H); 2.19 (t, J=7.9 Hz,2H); 2.86 to 2.98 (m, 2H); 3.01 to 3.16 (m, 3H); 3.35 (s, 3H); 3.52 (m,1H); 3.92 (dd, J=7.2 and 8.4 Hz, 1H); 3.99 (d, J=7.6 Hz, 1H); 4.09 (m,1H); 4.16 to 4.36 (m, 3H); 4.95 (broad d, J=4.6 Hz, 1H); 4.99 (d, J=5.1Hz, 1H); 5.03 (d, J=4.9 Hz, 1H); 7.27 to 7.37 (m, 3H); 7.41 (t, J=7.6Hz, 2H); 7.75 (split d, J=7.0 Hz, 2H); 7.85 (broad m, 1H); 7.89 (d,J=7.6 Hz, 2H); 8.23 (broad d, J=7.1 Hz, 1H). LCMS (A): ES m/z=700[M+H]⁺; m/z=744 [M−H+HCO₂H]⁻; t_(R)=1.22 min.

Compound 49:(S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-5-oxo-5-((((2R,3S,4S,5R,6R)-3,4,5-trihydroxy-6-methoxytetrahydro-2H-pyran-2-yl)methyl)amino)pentanoicAcid

To compound 48 (480 mg, 686 μmol), under magnetic stirring, was added a4M solution of HCl in 1,4-dioxane (30 mL). The reaction medium wasstirred at RT for 4 h. At this time, the medium was concentrated invacuo and purified by flash chromatography on 30 g of silica gel(elution DCM/MeOH/H₂O) to give 260 mg of compound 49 (59%).

RMN ¹H (400 MHz, δ in ppm, DMSO-d6): 0.83 (d, J=6.6 Hz, 3H); 0.85 (d,J=6.6 Hz, 3H); 1.69 to 1.90 (m, 2H); 2.01 to 2.15 (m, 3H); 2.86 to 2.97(m, 2H); 2.99 to 3.16 (m, 3H); 3.35 (s, 3H); 3.51 (m, 1H); 3.82 (m, 2H);3.99 (d, J=7.8 Hz, 1H); 4.20 to 4.33 (m, 3H); 4.95 (broad d, J=3.7 Hz,1H); 5.03 (d, J=4.9 Hz, 1H); 5.06 (broad d, J=4.6 Hz, 1H); 7.33 (m, 2H);7.40 (t, J=7.6 Hz, 2H); 7.52 (broad d, J=6.4 Hz, 1H); 7.61 (broad d,J=9.3 Hz, 1H); 7.73 (d, J=7.6 Hz, 1H); 7.76 (d, J=7.6 Hz, 1H); 7.89 (d,J=7.6 Hz, 2H); 8.10 (broad m, 1H). LCMS (A): ES m/z=642 [M−H]⁻; m/z=644[M+H]⁺; t_(R)=1.01 min.

Compound 50:(S)-2-((S)-2-amino-3-methylbutanamido)-N1-(4-((2R,3R)-3-((S)-1-((3S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13-en-16-yl)ethyl)oxiran-2-yl)benzyl)-N5-(((2R,3S,4S,5R,6R)-3,4,5-trihydroxy-6-methoxytetrahydro-2H-pyran-2-yl)methyl)pentanediamide

To a solution of(E)-(3S,10R,16S)-16-{(S)-1-[(2R,3R)-3-(4-aminomethyl-phenyl)-oxiranyl]-ethyl}-10-(3-chloro-4-methoxy-benzyl)-3-isobutyl-6,6-dimethyl-1,4-dioxa-8,11-diaza-cyclohexadec-13-ene-2,5,9,12-tetraone (thesynthesis of which was described in WO2011001052, compound 77, 110 mg,157 μmol) in DMF (6 mL) were added, under magnetic stirring, compound 49(122 mg, 189 μmol), HOBt (34 mg, 252 μmol) and EDC (43 μL, 236 μmol).The reaction medium was stirred at RT overnight. Then, 25 mg of compound49 and 25 μL of EDC were added to the medium, stirring was maintainedfor 1 h. At this time, piperidine (160 μL, 1.58 mmol) was added to themedium. After 2 h, the medium was concentrated in vacuo and purified byflash chromatography on 20 g of silica gel (gradient elution DCM/MeOH)to give 78 mg of compound 50 (45%).

RMN ¹H (500 MHz, δ in ppm, DMSO-d6): 0.76 (d, J=6.9 Hz, 3H); 0.79 (d,J=6.3 Hz, 6H); 0.87 (d, J=6.9 Hz, 3H); 1.00 (s, 3H); 1.04 (d, J=6.6 Hz,3H); 1.12 (s, 3H); 1.30 (m, 1H); 1.50 to 1.98 (m, 8H); 2.12 (m, 2H);2.26 (m, 1H); 2.68 (m, 2H); 2.86 to 3.14 (m, 9H); 3.32 (partially maskedm, 1H); 3.38 (s, 3H); 3.52 (m, 1H); 3.81 (s, 3H); 3.87 (s, 1H); 4.01 (d,J=7.4 Hz, 1H); 4.20 to 4.36 (m, 4H); 4.90 (m, 1H); 4.98 (broad d, J=3.6Hz, 1H); 5.02 (d, J=4.9 Hz, 1H); 5.06 (d, J=4.7 Hz, 1H); 5.10 (m, 1H);5.79 (d, J=15.6 Hz, 1H); 6.47 (m, 1H); 7.05 (d, J=8.5 Hz, 1H); 7.17(broad d, J=8.5 Hz, 1H); 7.21 to 7.26 (m, 5H); 7.29 (broad s, 1H); 7.90(m, 1H); 8.08 (m large, 1H); 8.37 (d, J=8.0 Hz, 1H); 8.48 (m, 1H). LCMS(A): ES m/z=551 [M+2H]²⁺; m/z=1099 [M−H]⁻; m/z=1101 [M+H]⁺; m/z=1145[M−H+HCO₂H]⁻; t_(R)=0.9 min.

Example 17:5-(((S)-1-(((S)-1-((4-((2R,3R)-3-((S)-1-((3S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13-en-16-yl)ethyl)oxiran-2-yl)benzyl)amino)-1,5-dioxo-5-((((2R,3S,4S,5R,6R)-3,4,5-trihydroxy-6-methoxytetrahydro-2H-pyran-2-yl)methyl)amino)pentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-5-oxopentanoicAcid

To a solution of compound 50 (77 mg, 69.9 μmol) in DMF (4 mL) was added,under magnetic stirring, glutaric anhydride (14 mg, 125.8 μmol). Thereaction medium was stirred at RT for 2 h. Then, the reaction medium wasconcentrated in vacuo and purified by flash chromatography on 10 g ofsilica gel (gradient elution DCM/MeOH) to give 54 mg of example 17(64%).

RMN ¹H (500 MHz, δ in ppm, DMSO-d6): 0.80 (d, J=6.0 Hz, 6H); 0.86 (d,J=6.6 Hz, 3H); 0.88 (d, J=6.6 Hz, 3H); 0.99 (s, 3H); 1.03 (d, J=6.6 Hz,3H); 1.12 (s, 3H); 1.34 (m, 1H); 1.53 to 1.85 (m, 6H); 1.90 to 2.33 (m,9H); 2.65 to 2.76 (m, 2H); 2.87 to 3.14 (m, 8H); 3.32 (partially maskedm, 1H); 3.35 (s, 3H); 3.53 (m, 1H); 3.81 (s, 3H); 3.87 (d, J=1.9 Hz,1H); 4.00 (d, J=7.9 Hz, 1H); 4.03 to 4.26 (m, 4H); 4.33 (m, 1H); 4.93(m, 1H); 5.02 (broad m, 3H); 5.10 (m, 1H); 5.80 (d, J=15.3 Hz, 1H); 6.46(ddd, J=3.5, 11.0 and 15.3 Hz, 1H); 7.05 (d, J=8.5 Hz, 1H); 7.18 (dd,J=2.2 and 8.5 Hz, 1H); 7.22 (d, J=8.2 Hz, 2H); 7.26 (d, J=8.2 Hz, 2H);7.30 (m, 2H); 7.98 to 8.65 (broad m, 5H). LCMS (A): ES m/z=608 [M+2H]²⁺;m/z=1213 [M−H]⁻; m/z=1215 [M+H]⁺; t_(R)=1.15 min.

Example 18: 2,5-dioxopyrrolidin-1-yl5-(((S)-1-(((S)-1-((4-((2R,3R)-3-((S)-1-((3S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13-en-16-yl)ethyl)oxiran-2-yl)benzyl)amino)-1,5-dioxo-5-((((2R,3S,4S,5R,6R)-3,4,5-trihydroxy-6-methoxytetrahydro-2H-pyran-2-yl)methyl)amino)pentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-5-oxopentanoate

Example 17 (45 mg, 37 μmol) was diluted in toluene and concentrated invacuo. Then, to a solution of example 17 in THF (5 mL), DCM (2 mL) andDMF (1 mL) were added, under magnetic stirring, DSC (11.4 mg, 44.4 μmol)and DIEA (19 μL, 112.6 μmol). The reaction medium was stirred at RTunder Ar for 3 h. Then, DMF and DSC were added to the medium untilcompletion of the reaction. After 3 h, the medium was diluted with water(5 mL), and extracted with MeTHF (3×10 mL). The combined organic phaseswere dried over MgSO₄, filtered and concentrated in vacuo. The mixturewas purified by flash chromatography on 4 g of silica gel (gradientelution DCM/iPrOH) to give 21 mg of example 18 (43%).

RMN ¹H (500 MHz, δ in ppm, DMSO-d6): 0.78 (d, J=6.0 Hz, 6H); 0.83 (d,J=6.9 Hz, 3H); 0.85 (d, J=6.9 Hz, 3H); 1.00 (s, 3H); 1.04 (d, J=6.3 Hz,3H); 1.12 (s, 3H); 1.30 (m, 1H); 1.52 to 1.63 (m, 2H); 1.75 to 2.02 (m,6H); 2.09 to 2.34 (m, 5H); 2.68 (m, 4H); 2.81 (s, 4H); 2.88 to 3.18 (m,7H); 3.32 (partially masked m, 1H); 3.38 (s, 3H); 3.51 (m, 1H); 3.81 (s,3H); 3.88 (s, 1H); 4.01 (d, J=7.7 Hz, 1H); 4.16 (dd, J=7.1 and 7.7 Hz,1H); 4.19 to 4.35 (m, 5H); 4.90 (m, 1H); 4.97 (d, J=4.1 Hz, 1H); 5.01(d, J=4.9 Hz, 1H); 5.06 (d, J=4.9 Hz, 1H); 5.10 (m, 1H); 5.79 (d, J=15.3Hz, 1H); 6.47 (m, 1H); 7.05 (d, J=8.5 Hz, 1H); 7.17 (d large, J=8.5 Hz,1H); 7.20 to 7.26 (m, 5H); 7.28 (broad s, 1H); 7.86 (broad t, J=5.6 Hz,1H); 7.91 (d, J=7.9 Hz, 1H); 8.08 (d, J=7.4 Hz, 1H); 8.33 (t, J=5.8 Hz,1H); 8.38 (d, J=8.2 Hz, 1H). LCMS (A): ES m/z=1310 [M−H]⁻; m/z=1356[M−H+HCO₂H]⁻; m/z=1312 [M+H]⁺; m/z=656.5 [M+2H]²⁺; t_(R)=1.2 min.

Example 19: hu2H11_R35-74-Ex18

The general method described previously was used for the preparation ofexample 19. 29.02 mg of hu2H11_R35-74 were reacted with 116.6 μL of a 10mM solution of example 18 in DMA (6 eq.) for 2 h 30. After purificationon Superdex 200 pg in buffer B pH 6.5+10% NMP, concentration on AmiconUltra-15, dilution in buffer B pH 6.5 to a final concentration of NMP at5% and filtration on 0.22 μm PVDF filter, 23.1 mg of example 19 wereobtained as a colorless limpid solution at a concentration of 2.2 mg/mLwith a DAR of 2.8 (HRMS), a monomeric purity of 99.9% and a global yieldof 78%. Free-drug level was above the threshold of 1%: the ADC wasconcentrated on Amicon Ultra-15, purified on Sephadex G25 in buffer B pH6.5+5% NMP and filtrated on 0.22 μm PVDF filter to provide 17.25 mg ofexample 19 as a colorless limpid solution at a concentration of 1.50mg/mL with a DAR of 2.55 (HRMS), a monomeric purity of 99.7% and aglobal yield of 59%.

SEC-HRMS: m/z=149405 (naked mAb); m/z=150602 (D1); m/z=151800 (D2);m/z=152997 (D3); m/z=154196 (D4); m/z=155393 (D5); m/z=156589 (D6);m/z=157791 (D7).

Synthesis of Example 20: glutaryl-Val-GlucamineGln-C52 Benzylic Amine

Compound 51: (S)-2,5-dioxopyrrolidin-1-yl2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanoate

To a solution of Fmoc-Val-OH (CAS number [68858-20-8], 5 g, 14.44 mmol)in THF (40 mL) were added NHS (1.71 g, 14.44 mmol) and DCC (2.98 g,14.44 mmol). The reaction medium was stirred for 5 h at RT, then 0.2equivalents of NHS and DCC were added and stirring was carried on for 4h at RT. The medium was filtered, the solid washed twice with THF (2×25mL) and the filtrate concentrated in vacuo to give 6.5 g of compound 51as a white meringue (quant.).

RMN ¹H (400 MHz, δ in ppm, DMSO-d6): 1.03 (d, J=6.9 Hz, 6H); 2.20 (m,1H); 2.81 (s, 4H); 4.18 to 4.39 (m, 4H); 7.32 (m, 2H); 7.42 (m, 2H);7.74 (m, 2H); 7.90 (d, J=7.6 Hz, 2H); 8.12 (d, J=8.5 Hz, 1H).

Compound 52:(S)-4-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-5-(tert-butoxy)-5-oxopentanoicAcid

To a solution of H-Glu-OtBu (CAS number [45120-30-7], 3.14 g, 15.16mmol) and sodium bicarbonate (1.40 g, 16.46 mmol) in H₂O (60 mL) wasadded a solution of compound 51 (6.3 g, 14.43 mmol) in THF (240 mL). Thereaction medium was stirred for 16 h at RT and concentrated in vacuo.The crude product was diluted in H₂O (700 mL) forming a gel that wasstirred for 15 min then extracted twice with Et₂O (200 mL). The aqueoussuspension was acidified to pH 3 with 5 N HCl and extracted with DCM(4×200 mL). The combined organic phases were dried over MgSO₄, filteredand concentrated in vacuo to give 7.98 g of compound 52 as a whitemeringue (quant.).

RMN ¹H (400 MHz, δ in ppm, DMSO-d6): 0.87 (d, J=6.9 Hz, 3H); 0.90 (d,J=6.9 Hz, 3H); 1.38 (s, 9H); 1.77 (m, 1H); 1.91 (m, 1H); 1.98 (m, 1H);2.28 (m, 2H); 3.90 (dd, J=7.1 and 9.1 Hz, 1H); 4.10 to 4.37 (m, 4H);7.32 (m, 2H); 7.38 (d, J=9.1 Hz, 1H); 7.41 (m, 2H); 7.73 (m, 2H); 7.89(d, J=7.6 Hz, 2H); 8.20 (d, J=7.5 Hz, 1H); 12.21 (broad m, 1H).

Compound 53: (S)-1-tert-butyl 5-(2,5-dioxopyrrolidin-1-yl)2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)pentanedioate

To a solution of compound 52 (500 mg, 953.1 μmol) in THF (30 mL) wereadded DIEA (320 μL, 1.9 mmol), DSC (498.3 mg, 1.91 mmol) and DMF (6 mL).The reaction medium was stirred at RT overnight then 30 mg of DSC wereadded to the medium and stirring was carried on 24 h. The reactionmedium was concentrated in vacuo, co-evaporated twice with toluene andpurified by flash chromatography on 50 g of silica gel (gradient elutionDCM/iPrOH) to give 506 mg of compound 53 as a white oil (85%).

RMN ¹H (400 MHz, δ in ppm, DMSO-d6): 0.89 (d, J=6.9 Hz, 3H); 0.91 (d,J=6.9 Hz, 3H); 1.39 (s, 9H); 1.75 to 2.11 (m, 3H); 2.73 (m, 2H); 2.80(s, 4H); 3.89 (dd, J=7.3 and 9.1 Hz, 1H); 4.20 to 4.38 (m, 4H); 7.32 (m,2H); 7.42 (m, 3H); 7.75 (m, 2H); 7.80 (m, 2H); 8.31 (d, J=7.5 Hz, 1H).

Compound 54:(2S,3R,4R,5R)-1-amino-6-((tert-butyldiphenylsilyl)oxy)hexane-2,3,4,5-tetraol

A suspension of D-glucamine (CAS number [488-43-7], 200 mg, 1.10 mmol)in DMF (2 mL) was cooled at 0° C. then were added imidazole (82.0 mg,1.19 mmol) and tert-butylchlorodiphenylsilane (334.4 mg, 1.19 mmol). Thereaction medium was stirred for 1 h at 0° C., quenched with H₂O (20 mL)and stirring carried on 10 min. The medium was extracted with EtOAc(3×50 mL), the combined organic phases were dried over MgSO₄, filtered,concentrated in vacuo, co-evaporated twice with toluene and purified byreverse phase flash chromatography on 31 g of C18-modified silica gel(gradient elution H₂O/MeCN) to give 218 mg of compound 54 as a whitefoam (47%).

RMN ¹H (400 MHz, δ in ppm, DMSO-d6): 1.00 (s, 9H); 2.62 (dd, J=6.3 and12.7 Hz, 1H); 2.74 (dd, J=4.5 and 12.7 Hz, 1H); 3.40 (partially maskedm, 1H); 3.49 (m, 1H); 3.55 to 3.72 (m, 3H); 3.88 (m, 1H); 4.48 (broad m,4H); 7.42 (m, 6H); 7.69 (m, 4H).

Compound 55: (S)-tert-butyl2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-5-(((2S,3R,4R,5R)-6-((tert-butyldiphenylsilyl)oxy)-2,3,4,5-tetrahydroxyhexyl)amino)-5-oxopentanoate

To a solution of compound 53 (300 mg, 482.6 μmol) in DMF (2 mL) wasadded a solution of compound 54 (214.6 mg, 511.5 μmol) in DMF (3 mL).The reaction medium was stirred for 1 h at RT, concentrated in vacuo,co-evaporated with toluene (3×) and purified by flash chromatography on25 g of silica gel (gradient elution DCM/MeOH/H₂O) to give 270 mg ofcompound 55 as a white foam (60%).

RMN ¹H (400 MHz, δ in ppm, DMSO-d6): 0.87 (d, J=6.9 Hz, 3H); 0.90 (d,J=6.9 Hz, 3H); 0.99 (s, 9H); 1.38 (s, 9H); 1.80 (m, 1H); 1.90 (m, 1H);1.98 (m, 1H); 2.17 (t, J=7.7 Hz, 2H); 3.03 (m, 1H); 3.26 (m, 1H); 3.49(m, 1H); 3.52 to 3.60 (m, 4H); 3.83 (m, 1H); 3.92 (dd, J=7.3 and 9.2 Hz,1H); 4.08 (m, 1H); 4.18 to 4.31 (m, 4H); 4.33 (d, J=6.4 Hz, 1H); 4.63(d, J=5.8 Hz, 1H); 4.78 (d, J=4.7 Hz, 1H); 7.31 (m, 2H); 7.36 (d, J=9.2Hz, 1H); 7.41 (m, 8H); 7.67 (m, 4H); 7.69 to 7.76 (m, 3H); 7.89 (m, 2H);8.23 (d, J=7.5 Hz, 1H).

Compound 56:(S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-5-(((2S,3R,4R,5R)-6-((tert-butyldiphenylsilyl)oxy)-2,3,4,5-tetrahydroxyhexyl)amino)-5-oxopentanoicAcid

Compound 55 was diluted in DCM forming a gel that was cooled at 0° C.before slowly adding, at 0° C., a 1:1 mixture of DCM/TFA. The reactionmedium was stirred for 30 min at 0° C. then 5 h at RT, concentrated invacuo and co-evaporated with toluene (3×) to give a white solid that wasgrinded up in iPr₂O, filtered, washed twice with Et₂O (5 mL), dried andfinally purified by flash chromatography on 15 g of silica gel (gradientelution DCM/MeOH/H₂O) to give 149 mg of compound 56 as a white solid(59%).

RMN ¹H (400 MHz, δ in ppm, DMSO-d6): 0.84 (d, J=6.8 Hz, 3H); 0.86 (d,J=6.8 Hz, 3H); 0.99 (s, 9H); 1.82 (m, 1H); 1.90 (m, 1H); 2.03 to 2.14(m, 3H); 3.00 (m, 1H); 3.25 (m, 1H); 3.48 (m, 1H); 3.65 (m, 4H); 3.82 to3.92 (m, 3H); 4.20 to 4.37 (m, 3H); 4.40 (d, J=6.0 Hz, 1H); 4.45 (d,J=6.0 Hz, 1H); 4.80 (m, 1H); 5.12 (m, 1H); 7.32 (m, 2H); 7.41 (m, 8H);7.55 (m, 2H); 7.65 to 7.82 (m, 7H); 7.89 (d, J=8.0 Hz, 2H). LCMS (A):m/z=868 [M−H]⁻; m/z=870 [M+H]⁺; t_(R)=1.55 min.

Compound 57:(S)-2-((S)-2-amino-3-methylbutanamido)-N5-((2S,3R,4R,5R)-6-((tert-butyldiphenylsilyl)oxy)-2,3,4,5-tetrahydroxyhexyl)-N1-(4-((2R,3R)-3-((S)-1-((3S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclo-hexadec-13-en-16-yl)ethyl)oxiran-2-yl)benzyl)-pentanediamide

To a solution of compound 56 (67.3 mg, 77.3 μmol) and(3S,10R,16S,E)-16-((S)-1-((2R,3R)-3-(4-(aminomethyl)phenyl)oxiran-2-yl)ethyl)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-1,4-dioxa-8,11-diazacyclohexadec-13-ene-2,5,9,12-tetraone(the synthesis of which was described in WO2011001052, compound 77, 45mg, 64.5 μmol) in DMF and DCM (10 mL) were added HOBt (11.5 mg, 85.1μmol) and EDC (11.4 mg, 70.9 μmol). The reaction medium was stirred for3 h at RT then quenched with H₂O (15 mL) and stirring was carried on 10min. The aqueous phase was extracted with DCM (3×20 mL), the combinedorganic phases were dried over MgSO₄, filtered, concentrated in vacuoand purified by flash chromatography on 5 g of silica gel (gradientelution DCM/MeOH) to give 64 mg of Fmoc-protected dipeptide-cryptophycinintermediate as a white solid (64%).

RMN ¹H (500 MHz, δ in ppm, DMSO-d6): 0.76 (d, J=6.8 Hz, 6H); 0.82 (d,J=7.0 Hz, 3H); 0.86 (d, J=7.0 Hz, 3H); 0.99 (s, 12H); 1.04 (d, J=7.1 Hz,3H); 1.10 (s, 3H); 1.30 (m, 1H); 1.55 (m, 2H); 1.80 (m, 2H); 1.90 (m,1H); 2.00 (m, 1H); 2.14 (m, 2H); 2.26 (m, 1H); 2.68 (m, 2H); 2.92 to3.08 (m, 4H); 3.22 to 3.38 (partially masked m, 2H); 3.49 (m, 1H); 3.58to 3.69 (m, 4H); 3.81 (s, 3H); 3.82 (m, 1H); 3.87 (d, J=2.0 Hz, 1H);3.91 (m, 1H); 4.18 to 4.38 (m, 9H); 4.68 (m, 1H); 4.82 (m, 1H); 4.90(dd, J=3.8 and 9.8 Hz, 1H); 5.10 (m, 1H); 5.79 (broad d, J=16.0 Hz, 1H);6.47 (ddd, J=4.7, 10.5 and 16.0 Hz, 1H); 7.05 (d, J=8.0 Hz, 1H); 7.17(dd, J=2.5 and 8.0 Hz, 1H); 7.20 to 7.27 (m, 5H); 7.29 (d, J=2.3 Hz,1H); 7.31 (t, J=7.8 Hz, 2H); 7.34 to 7.48 (m, 7H); 7.65 to 7.79 (m, 9H);7.90 (d, J=7.8 Hz, 2H); 8.10 (d, J=8.3 Hz, 1H); 8.38 (d, J=8.0 Hz, 1H);8.41 (t, J=6.5 Hz, 1H). LCMS (A): ES m/z=775 [M+2H]²⁺; m/z=1549 [M+H]⁺;m/z=1593 [M−H+HCO₂H]⁻; t_(R)=1.78 min.

To a solution of this intermediate (62 mg, 40.0 μmol) in DCM (8 mL) wasadded piperidine (59.8 μL, 600.0 μmol). The reaction medium was stirredfor 4 h at RT, concentrated in vacuo and purified by flashchromatography on 5 g of silica gel (gradient elution DCM/MeOH/H₂O) togive 49 mg of compound 57 as a white lacquer (92%).

Compound 58:(6R,7R,8R,9S,15S,18S)-15-((4-((2R,3R)-3-((S)-1-((3S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13-en-16-yl)ethyl)oxiran-2-yl)benzyl)carbamoyl)-6,7,8,9-tetrahydroxy-18-isopropyl-2,2-dimethyl-12,17,20-trioxo-3,3-diphenyl-4-oxa-11,16,19-triaza-3-silatetracosan-24-oicAcid

To a solution of compound 57 (23 mg, 17.3 μmol) in DCM (3 mL) was addeda solution of glutaric anhydride (2.22 mg, 19.1 μmol) in DCM (2 mL). Thereaction medium was stirred for 3 h at RT, partly concentrated in vacuo(down to 2 mL) and purified by flash chromatography on 5 g of silica gel(gradient elution DCM/MeOH/H₂O) to give 17 mg of compound 58 as a whitelacquer (68%).

RMN ¹H (500 MHz, δ in ppm, DMSO-d6): 0.80 (split d, J=6.8 Hz, 6H); 0.6(split d, J=7.0 Hz, 6H); 0.99 (s, 12H); 1.04 (d, J=7.1 Hz, 3H); 1.11 (s,3H); 1.32 (m, 1H); 1.52 to 1.60 (m, 2H); 1.72 to 2.00 (m, 7H); 2.08 (m,1H); 2.13 (m, 1H); 2.20 to 2.37 (m, 3H); 2.69 (m, 2H); 2.92 to 3.07 (m,4H); 3.20 (m, 1H); 3.30 (masked m, 1H); 3.45 (m, 1H); 3.55 to 3.70 (m,4H); 3.80 (s, 3H); 3.83 (m, 1H); 3.87 (d, J=2.3 Hz, 1H); 4.07 to 5.00(m, 11H); 5.10 (m, 1H); 5.80 (dd, J=2.0 and 15.8 Hz, 1H); 6.47 (m, 1H);7.04 (d, J=8.6 Hz, 1H); 7.18 (dd, J=2.4 and 8.6 Hz, 1H); 7.20 to 7.28(m, 5H); 7.29 (d, J=2.3 Hz, 1H); 7.41 (m, 6H); 7.68 (m, 4H); 8.05 (d,J=7.9 Hz, 1H; 8.23 (t, J=6.5 Hz, 1H); 8.30 to 8.60 (m, 3H). LCMS (A): ESm/z=721 [M+2H]²⁺; m/z=1439 [M−H]⁻; m/z=1441 [M+H]⁺; t_(R)=1.52 min.

Example 20:5-(((S)-1-(((S)-1-((4-((2R,3R)-3-((S)-1-((3S,10R,16S,E)-10-(3-chloro-4-methoxy-benzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13-en-16-yl)ethyl)oxiran-2-yl)benzyl)amino)-1,5-dioxo-5-(((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)-amino)pentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-5-oxopentanoicAcid

A solution of compound 57 (46 mg, 31.9 μmol) in THF (3 mL) was cooled at0° C. then was added, dropwise at 0° C., a 1 M TBAF solution in THF(35.1 μL, 35.1 μmol). The reaction medium was stirred for 2 h at 0° C.,quenched with H₂O (100 μL), stirred for 10 min and extracted with DCM (5mL). The organic phase was dried over MgSO₄, filtered and concentratedin vacuo: LCMS analysis showed the presence of residual startingmaterial. The crude product was dissolved in DCM (3 mL), the solutioncooled at 0° C. before adding, dropwise at 0° C., a 1 M TBAF solution inTHF (35.1 μL, 35.1 μmol). The reaction medium was stirred for 3 h at 0°C., quenched with H₂O (100 μL), stirred for 10 min and extracted withDCM (20 mL). The organic phase was dried over MgSO₄, filtered,concentrated in vacuo and purified by flash chromatography on 5 g ofsilica gel (gradient elution DCM/MeOH/H₂O to MeOH) then by reverse phaseflash chromatography on 3 g of C18-modified silica gel (gradient elutionH₂O/MeCN) to give 17 mg of example 20 as a white lacquer (44%).

RMN ¹H (500 MHz, δ in ppm, DMSO-d6): 0.80 (split d, J=6.8 Hz, 6H); 0.6(split d, J=7.0 Hz, 6H); 0.99 (s, 12H); 1.04 (d, J=7.1 Hz, 3H); 1.11 (s,3H); 1.32 (m, 1H); 1.52 to 1.60 (m, 2H); 1.72 to 2.00 (m, 7H); 2.08 (m,1H); 2.13 (m, 1H); 2.20 to 2.37 (m, 3H); 2.69 (m, 2H); 2.92 to 3.07 (m,4H); 3.20 (m, 1H); 3.30 (masked m, 1H); 3.45 (m, 1H); 3.55 to 3.70 (m,4H); 3.80 (s, 3H); 3.83 (m, 1H); 3.87 (d, J=2.3 Hz, 1H); 4.07 to 5.00(m, 11H); 5.10 (m, 1H); 5.80 (dd, J=2.0 and 15.8 Hz, 1H); 6.47 (m, 1H);7.04 (d, J=8.6 Hz, 1H); 7.18 (dd, J=2.4 and 8.6 Hz, 1H); 7.20 to 7.28(m, 5H); 7.29 (d, J=2.3 Hz, 1H); 7.41 (m, 6H); 7.68 (m, 4H); 8.05 (d,J=7.9 Hz, 1H); 8.23 (t, J=6.5 Hz, 1H); 8.30 to 8.60 (m, 3H). LCMS (A):ES m/z=602 [M+2H]²⁺; m/z=1201 [M−H]⁻; m/z=1203 [M+H]⁺; t_(R)=1.1 min.

Synthesis of Examples 21 to 23: glutaryl-Val-PEG4Lys-C52 Benzylic Amine,NHS Ester of glutaryl-Val-PEG4Lys-C52 Benzylic Amine and CorrespondingADC

Compound 59:(S)-20-((tert-butoxycarbonyl)amino)-14-oxo-2,5,8,11-tetraoxa-15-azahenicosan-21-oicAcid

To a solution of (S)-6-amino-2-((ter-butoxycarbonyl)amino)hexanoic acid(CAS number [13734-28-6], 73.9 mg, 300 μmol) in THF (300 μL) were added,under magnetic stirring, 2,5-dioxopyrrolidin-1-yl2,5,8,11-tetraoxatetradecan-14-oate (CAS number [622405-78-1], 100 mg,300 μmol), DIEA (53.3 μL, 300 μmol) and H₂O (100 μL). The reactionmedium was stirred at RT overnight then concentrated in vacuo. The crudemedium was purified by flash chromatography on 5 g of C18-grafted silicagel (gradient elution H₂O/CH₃CN) to give 81 mg of compound 59 (58%).LCMS (B): ES m/z=463 [M−H]⁻; m/z=465 [M+H]⁺; t_(R)=0.86 min.

Compound 60:(S)-20-amino-14-oxo-2,5,8,11-tetraoxa-15-azahenicosan-21-oic acidhydrochloride

To compound 59 (150.6 mg, 324.2 μmol), under magnetic stirring, wasadded a 4M solution of HCl in 1,4-dioxane (1 mL). The reaction mediumwas stirred at RT overnight then concentrated in vacuo. EtOAc was addedand the mixture was concentrated in vacuo to give 156 mg of compound 60(quant.).

LCMS (B): ES m/z=508 [M−H]⁻; m/z=510 [M+H]⁺; t_(R)=1.23 min.

Compound 61:(S)-20-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-14-oxo-2,5,8,11-tetraoxa-15-azahenicosan-21-oicAcid

To a solution of compound 60 (130 mg, 275 μmol) in water (0.5 mL) wasadded, under magnetic stirring, NaHCO₃ (55.6 mg, 661 μmol), followed by(S)-2,5-dioxopyrrolidin-1-yl2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanoate (CASnumber [130878-68-1], 144.4 mg, 330.8 μmol) in THF (1.5 mL). Thereaction medium was stirred at RT overnight. Then, NaHCO₃ (30 mg), THF(500 μL), H₂O (200 μL) and (S)-2,5-dioxopyrrolidin-1-yl2-((((9H-fluoren-9-yl)methoxy)carbonyl)-amino)-3-methylbutanoate (28 mg)were added to the reaction medium. After 3 h, the medium wasconcentrated in vacuo, then diluted with H₂O (20 mL) and ˜2-3 mL ofaqueous saturated Na₂CO₃. The aqueous phase was extracted twice withEtOAc (20 mL). The combined organic phases were washed with brine (20mL), dried over MgSO₄, filtered and concentrated in vacuo. The mixturewas purified by flash chromatography on 15 g of silica gel (elution85:1:0.5 v/v EtOAc/MeOH/H₂O). After concentration, the crude product wasdiluted with toluene and concentrated in vacuo to give 75.6 mg ofcompound 61 (40%).

RMN ¹H (400 MHz, δ in ppm, DMSO-d6): 0.85 (d, J=7.1 Hz, 3H); 0.87 (d,J=7.1 Hz, 3H); 1.21 to 1.40 (m, 4H); 1.50 to 1.73 (m, 2H); 2.01 (m, 1H);2.27 (t, J=6.6 Hz, 2H); 2.98 (m, 2H); 3.23 (s, 3H); 3.41 (m, 2H); 3.43to 3.52 (m, 10H); 3.57 (t, J=6.6 Hz, 2H); 3.88 (m, 1H); 4.01 (m, 1H);4.19 to 4.34 (m, 3H); 7.31 (m, 2H); 7.38 to 7.46 (m, 3H); 7.70 to 7.91(m, 6H); 12.51 (m, 1H). LCMS (A): ES m/z=684 [M−H]⁻; m/z=686 [M+H]⁺;t_(R)=1.2 min.

Compound 62:N—((S)-5-((S)-2-amino-3-methylbutanamido)-6-((4-((2R,3R)-3-((S)-1-((3S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13-en-16-yl)ethyl)oxiran-2-yl)benzyl)amino)-6-oxohexyl)-2,5,8,11-tetraoxatetradecan-14-amide

To compound 61 (70 mg, 102.1 μmol), under magnetic stirring and Ar, wasadded a solution of(E)-(3S,10R,16S)-16-{(S)-1-[(2R,3R)-3-(4-aminomethyl-phenyl)-oxiranyl]-ethyl}-10-(3-chloro-4-methoxy-benzyl)-3-isobutyl-6,6-dimethyl-1,4-dioxa-8,11-diaza-cyclohexadec-13-ene-2,5,9,12-tetraone (thesynthesis of which was described in WO2011001052, compound 77, 76.2 mg,109.1 μmol) in THF (3 mL), followed by HOBt (17.7 mg, 131 μmol) and EDC(23 μL, 131 μmol). The reaction medium was stirred at RT overnight.Then, piperidine (32.8 μL, 327.4 μmol) was added to the medium, andstirring was maintained for 4 h 30. At this time, the medium wasconcentrated in vacuo, diluted with DMA and purified by flashchromatography on 15 g of C18-grafted silica gel (gradient elutionH₂O/CH₃CN) to give 66 mg of compound 62(53%).

LCMS (B): ES m/z=572; m/z=1187 [M−H+HCO₂H]⁻; t_(R)=1.18 min.

Example 21:(20S,23S)-20-((4-((2R,3R)-3-((S)-1-((3S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13-en-16-yl)ethyl)oxiran-2-yl)benzyl)carbamoyl)-23-isopropyl-14,22,25-trioxo-2,5,8,11-tetraoxa-15,21,24-triazanonacosan-29-oicAcid

To a solution of compound 62 (66 mg, 57.7 μmol) in DMF (4 mL), undermagnetic stirring, was added glutaric anhydride (11.85 mg, 103.8 μmol).The reaction medium was stirred at RT for 2 h. At this time, the mediumwas concentrated in vacuo and purified by flash chromatography on 10 gof silica gel (gradient elution DCM/MeOH) to give 48.6 mg of example 21(80%).

RMN ¹H (500 MHz, δ in ppm, DMSO-d6): 0.81 (m, 6H); 0.85 (d, J=7.5 Hz,3H); 0.87 (d, J=7.5 Hz, 3H); 0.98 (s, 3H); 1.04 (d, J=7.1 Hz, 3H); 1.10(s, 3H); 1.16 to 1.44 (m, 5H); 1.55 to 1.79 (m, 7H); 1.90 to 2.10 (m,3H); 2.14 to 2.29 (m, 3H); 2.31 (t, J=6.7 Hz, 2H); 2.66 to 2.78 (m, 2H);2.85 to 2.99 (m, 4H); 3.05 (m, 1H); 3.23 (s, 3H); 3.32 (partially maskedm, 1H); 3.42 (m, 2H); 3.44 to 3.51 (m, 10H); 3.57 (m, 2H); 3.81 (s, 3H);3.88 (d, J=1.6 Hz, 1H); 4.04 to 4.25 (m, 4H); 4.35 (m, 1H); 4.94 (m,1H); 5.10 (m, 1H); 5.80 (d, J=15.0 Hz, 1H); 6.45 (ddd, J=4.0, 11.2 and15.0 Hz, 1H); 7.04 (d, J=8.6 Hz, 1H); 7.19 (dd, J=1.7 and 8.6 Hz, 1H);7.22 (d, J=8.4 Hz, 2H); 7.28 (d, J=8.4 Hz, 2H); 7.30 (d, J=1.7 Hz, 1H);7.32 (broad m, 1H); 7.75 to 9.42 (broad m, 5H); 12.06 (broad m, 1H).LCMS (A): ES m/z=629 [M+2H]²⁺; m/z=1255 [M−H]⁻; m/z=1257 [M+H]⁺;t_(R)=1.26 min.

Example 22: (20S,23S)-2,5-dioxopyrrolidin-1-yl20-((4-((2R,3R)-3-((S)-1-((3S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13-en-16-yl)ethyl)oxiran-2-yl)benzyl)carbamoyl)-23-isopropyl-14,22,25-trioxo-2,5,8,11-tetraoxa-15,21,24-triazanonacosan-29-oate

Toluene (10 mL) was added to example 21 (46 mg, 36.6 μmol) andconcentrated in vacuo, followed by dilution with THF (5 mL), DCM (2 mL)and DMF (200 μL). Then, DSC (11.5 mg, 43.9 μmol) was added, followed byDIEA (18.5 μL, 109.7 μmol). The reaction medium was stirred at RT for 4h. At this time, MeTHF (7 mL) and H₂O (3 mL) were added, the aqueousphase was extracted twice with MeTHF (5 mL). The combined organic phaseswere washed with H₂O (3 mL), dried over MgSO₄, filtered and concentratedin vacuo. The crude medium was purified by flash chromatography on 4 gof silica gel (gradient elution DCM/iPrOH) to give 33.3 mg of example 22(67%).

RMN ¹H (500 MHz, δ in ppm, DMSO-d6): 0.78 (d, J=6.6 Hz, 6H); 0.83 (d,J=7.1 Hz, 3H); 0.84 (d, J=7.1 Hz, 3H); 1.00 (s, 3H); 1.04 (d, J=6.9 Hz,3H); 1.12 (s, 3H); 1.20 to 1.43 (m, 5H); 1.50 to 1.69 (m, 4H); 1.76 to1.87 (m, 3H); 1.98 (m, 1H); 2.21 to 2.35 (m, 5H); 2.62 to 2.73 (m, 4H);2.81 (s, 4H); 2.94 to 3.04 (m, 5H); 3.23 (s, 3H); 3.32 (partially maskedm, 1H); 3.42 (m, 2H); 3.44 to 3.51 (m, 10H); 3.58 (t, J=6.4 Hz, 2H);3.81 (s, 3H); 3.87 (d, J=1.1 Hz, 1H); 4.17 (dd, J=7.3 and 7.8 Hz, 1H);4.21 to 4.31 (m, 4H); 4.90 (m, 1H); 5.10 (m, 1H); 5.79 (d, J=15.1 Hz,1H); 6.47 (ddd, J=3.5, 11.5 and 15.1 Hz, 1H); 7.05 (d, J=8.5 Hz, 1H);7.17 (dd, J=1.6 and 8.5 Hz, 1H); 7.20 to 7.25 (m, 5H); 7.28 (d, J=1.6Hz, 1H); 7.79 (t, J=5.7 Hz, 1H); 7.91 (d, J=8.5 Hz, 1H); 7.95 (d, J=8.3Hz, 1H); 8.37 (m, 2H). LCMS (C): ES m/z=677.5 [M+2H]²⁺; m/z=1354 [M+H]⁺;m/z=1399 [M−H+HCO₂H]⁻; t_(R)=4.41 min.

Example 23: hu2H11_R35-74-Ex22

The general method described previously was used for the preparation ofexample 23. 44.64 mg of hu2H11_R35-74 were reacted with 209.2 μL of a 10mM solution of example 22 in DMA (7 eq.) for 3 h 30. After purificationon Sephadex G25 in buffer B pH 6.5+5% NMP and concentration on AmiconUltra-15, 36.6 mg of example 23 were obtained as a colorless limpidsolution at a concentration of 1.83 mg/mL with a DAR of 3.6 (HRMS), amonomeric purity of 98.8% and a global yield of 82%. Free-drug level wasabove the threshold of 1%: the ADC was concentrated on Amicon Ultra-15,purified on Sephadex G25 in buffer B pH 6.5+5% NMP and filtrated on 0.22μm PVDF filter to provide 32.6 mg of example 23 as a colorless limpidsolution at a concentration of 1.63 mg/mL with a DAR of 3.3 (HRMS), amonomeric purity of 98.3% and a global yield of 73%.

SEC-HRMS: m/z=149411 (naked mAb); m/z=150649 (D1); m/z=151889 (D2);m/z=153129 (D3); m/z=154369 (D4); m/z=155608 (D5); m/z=156850 (D6);m/z=158091 (D7); m/z=159331 (D8); m/z=160583 (D9).

Synthesis of Examples 24 to 26: glutaryl-Val-PEG24Lys-C52 BenzylicAmine, NHS Ester of glutaryl-Val-PEG24Lys-C52 Benzylic Amine andCorresponding ADC

Compound 63:(S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-6-((tert-butoxycarbonyl)amino)hexanoicAcid

To a solution of H-Lys(Boc)-OH (CAS number [2418-95-3], 310 mg, 1.26mmol) and NaHCO₃ (105 mg, 1.25 mmol) in water (5 mL) and THF (5 mL) wasadded dropwise, under magnetic stirring, a solution of(S)-2,5-dioxopyrrolidin-1-yl2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanoate (CASnumber [68858-20-8], 310 mg, 1.15 mmol) in THF (15 mL). The reactionmedium was stirred at RT for 3 h. At this time, the reaction medium wasconcentrated in vacuo, then diluted with water (200 mL), acidified withQS of aqueous 5N HCl, and extracted with DCM (2×200 mL). The combinedorganic phases were dried over MgSO₄, filtered and concentrated in vacuoto afford 510 mg of compound 63 (53%).

RMN ¹H (400 MHz, δ in ppm, DMSO-d6): 0.85 (d, J=6.8 Hz, 3H); 0.87 (d,J=6.8 Hz, 3H); 1.20 to 1.39 (m, 4H); 1.34 (s, 9H); 1.48 to 1.73 (m, 2H);1.99 (m, 1H); 2.86 (m, 2H); 3.89 (dd, J=7.7 and 8.4 Hz, 1H); 4.08 (m,1H); 4.17 to 4.32 (m, 3H); 6.72 (broad m, 1H); 7.32 (m, 2H); 7.38 to7.46 (m, 3H); 7.74 (m, 2H); 7.89 (d, J=7.6 Hz, 2H); 8.00 (broad m, 1H);12.54 (broad s, 1H). LCMS (A): ES m/z=344; m/z=468; m/z=566 [M−H]⁻;m/z=568 [M+H]⁺; t_(R)=1.41 min.

Compound 64:(S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-6-aminohexanoicacid hydrochloride

To compound 63 (340 mg, 599 μmol) was added a solution of 4M HCl in1,4-dioxane, the reaction medium was stirred at RT for 1 h. At thistime, the reaction medium was concentrated in vacuo, followed byaddition of Et₂O and filtration. The white solid obtained was purifiedby flash chromatography on 15 g of silica gel (elution 12:3:0.5 v/v/vDCM/MeOH/NH₄OH) to give 190 mg of compound 64 (63%).

RMN ¹H (400 MHz, δ in ppm, DMSO-d6): 0.83 (d, J=6.6 Hz, 3H); 0.85 (d,J=6.6 Hz, 3H); 1.21 to 1.70 (m, 6H); 2.04 (m, 1H); 2.71 (t, J=7.1 Hz,2H); 3.84 (dd, J=6.7 and 8.7 Hz, 1H); 3.91 (m, 1H); 4.18 to 4.35 (m,3H); 7.22 (broad m, 3H); 7.33 (m, 2H); 7.42 (t, J=7.6 Hz, 2H); 7.54 (d,J=9.0 Hz, 1H); 7.69 (broad m, 1H); 7.74 (m, 2H); 7.89 (d, J=7.6 Hz, 2H).LCMS (A): ES m/z=244; m/z=466 [M−H]⁻; m/z=468 [M+H]⁺; t_(R)=0.81 min.

Compound 65:(S)-80-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-74-oxo-2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71-tetracosaoxa-75-azahenoctacontan-81-oic Acid

To a solution of compound 64 (130 mg, 258.0 μmol) in THF (20 mL) wereadded a solution of sodium bicarbonate (50 mg, 589.2 μmol) in H₂O (8 mL)and MEO-DPEG(24)-NHS (CAS number [756525-94-7], 400 mg, 329.4 μmol). Thereaction medium was stirred for 20 h at RT, partly concentrated in vacuothen diluted with H₂O (10 mL), acidified to pH 3 with IR-120 (H)Amberlite resin (CAS number [78922-04-0]), filtered, concentrated invacuo and purified by three consecutive flash chromatographies on 40 g,25 g and 12 g of silica gel (gradient elution DCM/MeOH/H₂O) to give 214mg of compound 65 as a white lacquer (53%).

RMN ¹H (400 MHz, δ in ppm, DMSO-d6): 0.86 (d, J=7.0 Hz, 3H); 0.89 (d,J=7.0 Hz, 3H); 1.22 to 1.40 (m, 4H); 1.58 (m, 1H); 1.67 (m, 1H); 1.99(m, 1H); 2.28 (t, J=6.6 Hz, 2H); 2.99 (m, 2H); 3.23 (s, 3H); 3.40 to3.70 (m, 92H); 3.56 (t, J=6.6 Hz, 2H); 3.90 (m, 1H); 4.08 (broad m, 1H);4.19 to 4.31 (m, 3H); 7.31 (broad t, J=7.8 Hz, 2H); 7.41 (broad t, J=7.8Hz, 3H); 7.73 (t, J=7.8 Hz, 2H); 7.76 (m, 1H); 7.89 (d, J=7.8 Hz, 2H);8.00 (m, 1H); 12.53 (m, 1H). LCMS (D): ES m/z=794 [M+H+Na]²⁺; m/z=1567[M+H]+; t_(R)=2.9 min.

Compound 66:N—((S)-5-((S)-2-amino-3-methylbutanamido)-6-((4-((2R,3R)-3-((S)-1-((3S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4dioxa-8,11-diazacyclohexadec-13-en-16-yl)ethyl)oxiran-2-yl)benzyl)-amino)-6-oxohexyl)-2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71-tetracosaoxatetraheptacontan-74-amide

To a solution of(E)-(3S,10R,16S)-16-{(S)-1-[(2R,3R)-3-(4-aminomethyl-phenyl)-oxiranyl]-ethyl}-10-(3-chloro-4-methoxy-benzyl)-3-isobutyl-6,6-dimethyl-1,4-dioxa-8,11-diaza-cyclohexadec-13-ene-2,5,9,12-tetraone (thesynthesis of which was described in WO2011001052, compound 77, 71 mg,101.7 μmol) in DMF (5 mL) were added a solution of compound 65 (214 mg,136.6 μmol) in DMF (5 mL), HOBt (22 mg, 156.3 μmol) and EDC (27 μL,152.5 μmol). The reaction medium was stirred for 3 h at RT before addingpiperidine (91 μL, 915.3 μmol). Stirring was carried on for 1 h 30 at RTthen the reaction medium was concentrated in vacuo and purified by twoconsecutive flash chromatographies on 25 g and 12 g of silica gel(gradient elution DCM/MeOH) to give 58 mg of compound 66 as a colorlesslacquer (28%).

RMN ¹H (500 MHz, δ in ppm, DMSO-d6): 0.76 (d, J=7.0 Hz, 3H); 0.78 (d,J=6.8 Hz, 6H); 0.88 (d, J=7.0 Hz, 3H); 0.99 (s, 3H); 1.04 (d, J=7.1 Hz,3H); 1.12 (s, 3H); 1.25 to 1.41 (m, 5H); 1.51 to 1.70 (m, 4H); 1.80 (m,1H); 1.93 (m, 1H); 2.25 (m, 1H); 2.28 (t, J=6.7 Hz, 2H); 2.55 (broad m,2H); 2.68 (m, 2H); 2.99 (m, 5H); 3.23 (s, 3H); 3.32 (masked m, 1H); 3.40to 3.55 (m, 92H); 3.58 (t, J=6.7 Hz, 2H); 3.80 (s, 3H); 3.88 (d, J=2.3Hz, 1H); 4.20 to 4.35 (m, 4H); 4.91 (dd, J=3.8 and 9.8 Hz, 1H); 5.10 (m,1H); 5.80 (dd, J=2.0 and 15.8 Hz, 1H); 6.46 (ddd, J=4.7, 10.5 and 15.8Hz, 1H); 7.05 (d, J=8.6 Hz, 1H); 7.17 (dd, J=2.4 et 8.6 Hz, 1H); 7.22(m, 5H); 7.29 (d, J=2.4 Hz, 1H); 7.79 (t, J=6.7 Hz, 1H); 8.00 (d, J=8.8Hz, 1H); 8.36 (d, J=8.0 Hz, 1H); 8.47 (t, J=6.2 Hz, 1H). LCMS (D): ESm/z=1013 [M+2H]²⁺; m/z=2025 [M+H]+; t_(R)=2.64/2.62 min (85/15 isomermixture).

Example 24:(80S,83S)-80-((4-((2R,3R)-3-((S)-1-((3S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13-en-16-yl)ethyl)oxiran-2-yl)benzyl)carbamoyl)-83-isopropyl-74,82,85-trioxo-2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71-tetracosaoxa-75,81,84-triazanonaoctacontan-89-oic Acid

To a solution of compound 66 (58 mg, 28.7 μmol) in DCM (7 mL) was addedglutaric anhydride (5.85 mg, 48.7 μmol). The reaction medium was stirredfor 2 h at RT, concentrated in vacuo and purified by flashchromatography on 4 g of silica gel (gradient elution DCM/MeOH/H2O) togive 35 mg of example 24 as a white lacquer (57%).

RMN ¹H (500 MHz, δ in ppm, DMSO-d6): 0.78 (d, J=6.8 Hz, 6H); 0.82 (d,J=7.0 Hz, 3H); 0.84 (d, J=7.0 Hz, 3H); 1.00 (s, 3H); 1.04 (d, J=7.1 Hz,3H); 1.11 (s, 3H); 1.18 to 1.41 (m, 5H); 1.50 to 1.75 (m, 6H); 1.80 (m,1H); 1.98 (m, 1H); 2.15 to 2.27 (m, 5H); 2.30 (t, J=6.7 Hz, 2H); 2.69(m, 2H); 2.98 (m, 5H); 3.23 (s, 3H); 3.32 (masked m, 1H); 3.40 to 3.65(m, 94H); 3.80 (s, 3H); 3.87 (d, J=2.3 Hz, 1H); 4.14 (m, 1H); 4.20 to4.33 (m, 4H); 4.90 (dd, J=3.8 and 9.8 Hz, 1H); 5.10 (m, 1H); 5.79 (d,J=15.8 Hz, 1H); 6.47 (ddd, J=4.7, 10.5 and 15.8 Hz, 1H); 7.05 (d, J=8.6Hz, 1H); 7.16 (dd, J=2.4 and 8.6 Hz, 1H); 7.24 (m, 5H); 7.29 (d, J=2.4Hz, 1H); 7.80 (t, J=6.0 Hz, 1H); 7.86 (d, J=8.8 Hz, 1H); 7.92 (d, J=8.0Hz, 1H); 8.37 (m, 2H); 12.10 (broad m, 1H). LCMS (D): ES m/z=1069.5[M+2H]²⁺; m/z=2138 [M+H]+; t_(R)=2.96/2.93 min (85/15 isomer mixture).

Example 25: (80S,83S)-2,5-dioxopyrrolidin-1-yl80-((4-((2R,3R)-3-((S)-1-((3S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclo-hexadec-13-en-16-yl)ethyl)oxiran-2-yl)benzyl)carbamoyl)-83-isopropyl-74,82,85-trioxo-2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71-tetracosaoxa-75,81,84-triazanona-octacontan-89-oate

To a solution of example 24 (35 mg, 16.4 μmol) in THF (3 mL) were addedDSC (4.28 mg, 16.4 μmol) and DIEA (2.7 μL, 16.4 μmol). The reactionmedium was stirred for 20 h at RT, concentrated in vacuo and purified byflash chromatography on 4 g of silica gel (gradient elution DCM/iPrOH)to give 12.0 mg of example 25 as a white lacquer (33%).

RMN ¹H (500 MHz, δ in ppm, DMSO-d6): 0.78 (d, J=6.8 Hz, 6H); 0.82 (d,J=7.0 Hz, 3H); 0.84 (d, J=7.0 Hz, 3H); 1.00 (s, 3H); 1.04 (d, J=7.1 Hz,3H); 1.12 (s, 3H); 1.15 to 1.40 (m, 5H); 1.50 to 1.71 (m, 4H); 1.75 to1.86 (m, 3H); 1.97 (m, 1H); 2.18 to 2.32 (m, 5H); 2.67 (m, 4H); 2.80 (s,4H); 2.98 (m, 5H); 3.22 (s, 3H); 3.32 (masked m, 1H); 3.40 to 3.77 (m,94H); 3.80 (s, 3H); 3.88 (d, J=2.3 Hz, 1H); 4.16 (m, 1H); 4.20 to 4.32(m, 4H); 4.90 (dd, J=3.8 and 9.8 Hz, 1H); 5.10 (m, 1H); 5.78 (dd, J=2.0and 15.8 Hz, 1H); 6.47 (ddd, J=4.7, 10.5 and 15.8 Hz, 1H); 7.05 (d,J=8.6 Hz, 1H); 7.16 (dd, J=2.4 and 8.6 Hz, 1H); 7.24 (m, 5H); 7.29 (d,J=2.4 Hz, 1H); 7.79 (t, J=6.0 Hz, 1H); 7.90 (d, J=8.8 Hz, 1H); 7.94 (d,J=8.0 Hz, 1H); 8.36 (m, 2H). LCMS (D): ES m/z=746; m/z=2235 [M+H]+;t_(R)=3.08/3.06 min (89/11 isomer mixture).

Example 26: hu2H11_R35-74-Ex25

The general method described previously was used for the preparation ofexample 26. 55.8 mg of hu2H11_R35-74 were reacted with 261.6 μL of a 10mM solution of example 25 in DMA (7 eq.) for 3 h 15 then were added 131μL of a 10 mM solution of example 25 in DMA (3.5 eq.) for 5 h 30. Afterstorage overnight at 4° C., purification on Superdex 200 pg in buffer BpH 6.5+20% NMP, concentration on Amicon Ultra-15, buffer exchange onSephadex G25 in buffer B pH 6.5+5% NMP, concentration on Amicon Ultra-15and filtration on 0.22 μm PVDF filter, 33.35 mg of example 26 wereobtained as a colorless limpid solution at a concentration of 2.9 mg/mLwith a DAR of 3.9 (HRMS), a monomeric purity of 100% and a global yieldof 60%.

SEC-HRMS: m/z=151487 (D1); m/z=153615 (D2); m/z=155740 (D3); m/z=157863(D4); m/z=159981 (D5); m/z=162112 (D6); m/z=164240 (D7); m/z=166356(D8).

Synthesis of Examples 27 to 29: glutaryl-Val-PEG24Lys-aza-cryptoBenzylic Amine, NHS Ester of glutaryl-Val-PEG24Lys-aza-crypto BenzylicAmine and Corresponding ADC

Compound 67:

N—((S)-5-((S)-2-amino-3-methylbutanamido)-6-((4-((2R,3R)-3-((S)-1-((3S,7S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-6,6,7-trimethyl-3-neopentyl-2,5,9,12-tetraoxo-1-oxa-4,8,11-triazacyclohexadec-13-en-16-yl)ethyl)oxiran-2-yl)benzyl)amino)-6-oxohexyl)-2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71-tetracosaoxatetraheptacontan-74-amide

To a solution of(3S,7S,10R,16S,E)-16-((S)-1-((2R,3R)-3-(4-(aminomethyl)phenyl)-oxiran-2-yl)ethyl)-10-(3-chloro-4-methoxybenzyl)-6,6,7-trimethyl-3-neopentyl-1-oxa-4,8,11-triazacyclo-hexadec-13-ene-2,5,9,12-tetraone(that can be synthesized as described in PCT/EP2016/076603 starting frommethyl (3S)-3-amino-2,2-dimethylbutanoate, [MFCD09256689], 75 mg, 103.4μmol) in DMF (5 mL) were added a solution of compound 65 (211 mg, 134.7μmol) in DMF (5 mL), HOBt (21 mg, 155.4 μmol) and EDC (20 μL 123.0μmol). The reaction medium was stirred for 4 h at RT before addingpiperidine (95 μL, 961.9 μmol); stirring was carried on for 2 h at RTthen the reaction medium was concentrated in vacuo and purified by flashchromatography on 25 g of silica gel (gradient elution DCM/MeOH) to give132 mg of compound 67 as a colorless oil (62%).

RMN ¹H (500 MHz, δ in ppm, DMSO-d6): 0.76 (d, J=7.0 Hz, 3H); 0.87 (m,15H); 1.00 (s, 3H); 1.03 (d, J=7.1 Hz, 3H); 1.15 to 1.40 (m, 5H); 1.18(s, 3H); 1.46 to 2.00 (m, 8H); 2.25 (m, 1H); 2.28 (t, J=6.7 Hz, 2H);2.60 (m, 1H); 2.69 (dd, J=11.2 and 14.5 Hz, 1H); 2.91 (dd, J=2.3 and 7.5Hz, 1H); 2.92 to 3.04 (m, 4H); 3.23 (s, 3H); 3.34 to 3.77 (m, 95H); 3.80(s, 3H); 3.88 (d, J=2.3 Hz, 1H); 4.08 (m, 2H); 4.20 to 4.34 (m, 3H);5.04 (m, 1H); 5.78 (dd, J=2.0 and 15.8 Hz, 1H); 6.44 (ddd, J=4.7, 10.5and 15.8 Hz, 1H); 7.02 (d, J=8.6 Hz, 1H); 7.17 to 7.25 (m, 5H); 7.32 (d,J=2.4 Hz, 1H); 7.78 (t, J=6.0 Hz, 1H); 7.85 (d, J=8.0 Hz, 1H); 7.92 (d,J=7.0 Hz, 1H); 8.00 (d, J=8.8 Hz, 1H); 8.40 (t, J=7.5 Hz, 1H); 8.46 (t,J=6.0 Hz, 1H). LCMS (D): ES m/z=2051 [M+H]⁺; t_(R)=2.57/2.55 min (isomermixture 95/5).

Example 27:(80S,83S)-80-((4-((2R,3R)-3-((S)-1-((3S,7S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-6,6,7-trimethyl-3-neopentyl-2,5,9,12-tetraoxo-1-oxa-4,8,11-triazacyclohexadec-13-en-16-yl)ethyl)oxiran-2-yl)benzyl)carbamoyl)-83-isopropyl-74,82,85-trioxo-2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71-tetracosaoxa-75,81,84-triazanonaoctacontan-89-oic Acid

To a solution of compound 67 (132 mg, 64.3 μmol) in DCM (10 mL) wasadded glutaric anhydride (9 mg, 78.9 μmol). The reaction medium wasstirred for 2 h at RT, concentrated in vacuo and purified by twoconsecutive flash chromatographies on 4 g and 12 g of silica gel(gradient elution DCM/MeOH/H₂O) to give 71 mg of example 27 as a whitelacquer (51%).

RMN ¹H (500 MHz, δ in ppm, DMSO-d6): 0.80 to 0.90 (m, 18H); 1.00 (s,3H); 1.03 (d, J=7.1 Hz, 3H); 1.15 to 1.42 (m, 5H); 1.18 (s, 3H); 1.51 to1.74 (m, 4H); 1.80 (m, 1H); 1.98 (m, 2H); 2.15 to 2.32 (m, 7H); 2.60 (m,1H); 2.70 (dd, J=11.2 and 14.5 Hz, 1H); 2.91 (dd, J=2.3 and 7.5 Hz, 1H);2.95 (dd, J=3.4 and 14.5 Hz, 1H); 3.00 (m, 2H); 3.24 (s, 3H); 3.40 to3.67 (m, 95H); 3.80 (s, 3H); 3.90 (d, J=2.3 Hz, 1H); 4.00 to 4.34 (m,6H); 5.04 (m, 1H); 5.80 (dd, J=2.0 and 15.8 Hz, 1H); 6.44 (ddd, J=4.7,10.5 and 15.8 Hz, 1H); 7.02 (d, J=8.6 Hz, 1H); 7.17 to 7.25 (m, 5H);7.32 (d, J=2.4 Hz, 1H); 7.80 (t, J=6.0 Hz, 1H); 7.86 (d, J=8.6 Hz, 2H);7.92 (t, J=6.5 Hz, 2H); 8.37 (t, J=6.0 Hz, 1H); 8.41 (t, J=7.5 Hz, 1H);12.00 (m, 1H). LCMS (D): ES m/z=722; m/z=2165 [M+H]⁺; t_(R)=2.95/2.92min (isomer mixture 95/5).

Example 28: 2,5-dioxopyrrolidin-1-yl(80S,83S)-80-((4-((2R,3R)-3-((S)-1-((3S,7S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-6,6,7-trimethyl-3-neopentyl-2,5,9,12-tetraoxo-1-oxa-4,8,11-triaza-cyclohexadec-13-en-16-yl)ethyl)oxiran-2-yl)benzyl)carbamoyl)-83-isopropyl-74,82,85-trioxo-2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71-tetracosaoxa-75,81,84-triazanonaoctacontan-89-oate

To a solution of example 27 (33 mg, 15.2 μmol) in THF (3 mL) were addedDSC (4 mg, 15.6 μmol) and DIEA (2.55 μL, 15.4 μmol). The reaction mediumwas stirred for 20 h at RT then were added DSC (1 mg, 3.9 μmol) and DIEA(1 μL, 6.0 μmol).

The reaction medium was stirred for 2 h at RT, concentrated in vacuo andpurified by flash chromatography on 4 g of silica gel (gradient elutionDCM/iPrOH/MeCN) to give 11 mg of example 28 as a white lacquer (32%).

RMN ¹H (500 MHz, δ in ppm, DMSO-d6): 0.80 to 0.90 (m, 18H); 1.00 (s,3H); 1.03 (d, J=7.1 Hz, 3H); 1.12 to 1.42 (m, 5H); 1.19 (s, 3H); 1.55(m, 1H); 1.65 (m, 1H); 1.82 (m, 3H); 1.96 (m, 2H); 2.28 (m, 3H); 2.67(m, 4H); 2.80 (s, 4H); 2.91 (dd, J=2.3 and Hz, 1H); 2.95 (dd, J=3.4 and14.5 Hz, 1H); 3.00 (m, 2H); 3.23 (s, 3H); 3.35 to 3.67 (m, 95H); 3.80(s, 3H); 3.90 (d, J=2.3 Hz, 1H); 4.03 to 4.32 (m, 6H); 5.03 (m, 1H);5.90 (dd, J=2.0 and 15.8 Hz, 1H); 6.44 (ddd, J=4.7, 10.5 and 15.8 Hz,1H); 7.03 (d, J=8.6 Hz, 1H); 7.17 to 7.25 (m, 5H); 7.32 (d, J=2.4 Hz,1H); 7.79 (t, J=6.0 Hz, 1H); 7.85 (d, J=8.7 Hz, 1H); 7.90 (d, J=8.7 Hz,1H); 7.93 (m, 2H); 8.35 (t, J=6.5 Hz, 1H); 8.40 (d, J=7.1 Hz, 1H).

Example 29: hu2H11_R35-74-Ex28

The general method described previously was used for the preparation ofexample 29. 44.64 mg of hu2H11_R35-74 were reacted with 210 μL of a 10mM solution of example 28 in DMA (7 eq.) for 2 h then were successivelyadded 210 μL of a 10 mM solution of example 28 in DMA (7 eq.) for 2 h 30and 90 μL of a 10 mM solution of example 28 in DMA (3 eq.) for 2 h.After storage overnight at 4° C., purification on Superdex 200 pg inbuffer B pH 6.5+20% NMP, concentration on Amicon Ultra-15, bufferexchange on Sephadex G25 in buffer B pH 6.5+5% NMP and filtration on0.22 μm PVDF filter, 30.1 mg of example 29 were obtained as a colorlesslimpid solution at a concentration of 2.51 mg/mL with a DAR of 4 (HRMS),a monomeric purity of 98.4% and a global yield of 67%.

SEC-HRMS: m/z=151502 (D1); m/z=153649 (D2); m/z=155798 (D3); m/z=157939(D4); m/z=160100 (D5); m/z=162262 (D6).

Synthesis of Examples 30 to 32: glutaryl-Val-GlucuronicLys-C52 BenzylicAmine, NHS Ester of glutaryl-Val-GlucuronicLys-C52 Benzylic Amine andCorresponding ADC

Compound 68:(S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-6-((2S,3S,4S,5R,6R)-3,4,5-trihydroxy-6-methoxytetrahydro-2H-pyran-2-carboxamido)hexanoicAcid

In a round bottom flask, under magnetic stirring,1-O-methyl-β-D-glucuronic acid, sodium salt (CAS number [58189-74-5],100 mg, 434 μmol) was introduced, followed by water (5 mL) and QS ofAmberlite (CAS number [9037-24-5]) to reach pH 2, the medium wasfiltered.

In a round bottom flask, under magnetic stirring, NaHCO₃ (43 mg, 511μmol) in water (5 mL) was added to a solution compound 64 (50 mg, 99μmol) in THF (4 mL); the reaction medium was stirred at RT for 5 min.Then, the glucuronic acid solution described above was added to thereaction medium, followed by4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chlorideN-hydrate (CAS number [3945-69-5], 67 mg, 242 μmol). The reaction mediumwas stirred at RT overnight, then concentrated in vacuo. The crudemedium was purified by flash chromatography on 5 g of silica gel(gradient elution DCM/MeOH/H₂O) to give 23 mg of compound 68 (35%).

RMN ¹H (400 MHz, δ in ppm, DMSO-d6): 0.84 (d, J=6.7 Hz, 3H); 0.86 (d,J=6.7 Hz, 3H); 1.13 to 1.42 (m, 4H); 1.52 to 1.72 (m, 2H); 2.05 (m, 1H);2.96 to 3.08 (m, 2H); 3.14 (m, 1H); 3.30 (partially masked m, 1H); 3.38(s, 3H); 3.53 (m, 1H); 3.78 to 3.97 (m, 2H); 4.09 (dd, J=2.3 and 7.8 Hz,1H); 4.19 to 4.34 (m, 3H); 5.01 to 5.08 (broad m, 1H); 5.13 (broad m,1H); 5.27 (m, 1H); 7.33 (m, 2H); 7.42 (t, J=7.6 Hz, 2H); 7.52 to 7.62(broad m, 2H); 7.75 (m, 2H); 7.89 (d, J=7.6 Hz, 2H); 7.93 (broad m, 1H);12.00 (m, 1H). LCMS (A): ES m/z=656 [M−H]⁻; m/z=658 [M+H]⁺; t_(R)=1.05min.

Compound 69:(2S,3S,4S,5R,6R)—N—((S)-5-((S)-2-amino-3-methylbutanamido)-6-((4-((2R,3R)-3-((S)-1-((3S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13-en-16-yl)ethyl)-oxiran-2-yl)benzyl)amino)-6-oxohexyl)-3,4,5-trihydroxy-6-methoxytetrahydro-2H-pyran-2-carboxamide

To a solution of(E)-(3S,10R,16S)-16-{(S)-1-[(2R,3R)-3-(4-aminomethyl-phenyl)-oxiranyl]-ethyl}-10-(3-chloro-4-methoxy-benzyl)-3-isobutyl-6,6-dimethyl-1,4-dioxa-8,11-diaza-cyclohexadec-13-ene-2,5,9,12-tetraone (thesynthesis of which was described in WO2011001052, compound 77, 90 mg,129 μmol) was added, under magnetic stirring, a solution of compound 68(135 mg, 205 μmol) in DMF (5 mL), followed by HOBt (30 mg, 222 μmol) andEDC (36 μL, 203 μmol). The reaction medium was stirred at RT for 1 h. Atthis time, DMA (1 mL) was added, and stirring was maintained overnight.Then, EDC (20 μL) was added and the reaction medium was stirred for 2more hours. Piperidine (12.9 μL, 129 μmol) was then added to the medium.After 1 h30 of stirring, the reaction medium was concentrated in vacuoand purified by 3 consecutive flash chromatographies on silica gel(isocratic elution 40:5:0.5 v/v/v DCM/MeOH/H₂O and 12:3:0.5 v/v/vDCM/MeOH/NH₄OH) to give 47 mg of compound 69 (33%).

RMN ¹H (500 MHz, δ in ppm, DMSO-d6): 0.77 to 0.81 (m, 9H); 0.88 (d,J=6.6 Hz, 3H); 1.00 (s, 3H); 1.04 (d, J=6.6 Hz, 3H); 1.12 (s, 3H); 1.20to 1.71 (m, 9H); 1.80 (m, 1H); 1.93 (m, 1H); 2.28 (m, 1H); 2.62 to 2.73(m, 2H); 2.93 to 3.14 (m, 9H); 3.28 to 3.36 (partially masked m, 2H);3.39 (s, 3H); 3.53 (d, J=9.5 Hz, 1H); 3.81 (s, 3H); 3.87 (d, J=1.7 Hz,1H); 4.09 (m, 1H); 4.19 to 4.36 (m, 4H); 4.91 (m, 1H); 5.00 to 5.15 (m,4H); 5.79 (d, J=15.9 Hz, 1H); 6.48 (m, 1H); 7.05 (d, J=8.6 Hz, 1H); 7.17(dd, J=2.1 and 8.6 Hz, 1H); 7.20 to 7.25 (m, 5H); 7.28 (d, J=2.1 Hz,1H); 7.92 (broad t, J=5.9 Hz, 1H); 8.02 (broad m, 1H); 8.35 (d, J=7.8Hz, 1H); 8.46 (broad t, J=6.2 Hz, 1H). LCMS (A): ES m/z=558 [M+2H]²⁺;m/z=1113 [M−H]⁻; m/z=1115 [M+H]⁺; m/z=1159 [M−H+HCO₂H]⁻; t_(R)=0.9 min.

Example 30:5-(((S)-1-(((S)-1-((4-((2R,3R)-3-((S)-1-((3S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13-en-16-yl)ethyl)oxiran-2-yl)benzyl)amino)-1-oxo-6-((2S,3S,4S,5R,6R)-3,4,5-trihydroxy-6-methoxytetrahydro-2H-pyran-2-carboxamido)hexan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-5-oxopentanoicAcid

To compound 69 (47 mg, 42.1 μmol) was added DMF (4 mL) and, undermagnetic stirring, glutaric anhydride (8.5 mg, 74.5 μmol); the reactionmedium was stirred at RT for 2 h. At this time, the crude medium wasconcentrated in vacuo, and purified by 2 consecutive flashchromatographies on silica gel (gradient elution DCM/MeOH/H₂O andDCM/MeOH/NH₄OH) to give 17 mg of example 30 (33%).

RMN ¹H (500 MHz, δ in ppm, DMSO-d6): 0.81 (split d, J=6.4 Hz, 6H); 0.84(d, J=6.6 Hz, 3H); 0.86 (d, J=6.6 Hz, 3H); 0.98 (s, 3H); 1.04 (d, J=6.6Hz, 3H); 1.11 (s, 3H); 1.17 to 2.32 (broad m, 18H); 2.66 to 2.74 (m,2H); 2.91 to 3.03 (m, 5H); 3.10 (m large, 1H); 3.18 (t, J=8.8 Hz, 1H);3.28 to 3.36 (partially masked m, 2H); 3.37 (s, 3H); 3.60 (m, 1H); 3.81(s, 3H); 3.87 (d, J=1.6 Hz, 1H); 4.07 (m, 2H); 4.14 to 4.26 (m, 3H);4.31 (m, 1H); 4.92 (m, 1H); 5.06 to 5.13 (m, 2H); 5.35 (m, 2H); 5.80 (d,J=15.3 Hz, 1H); 6.45 (ddd, J=3.6, 11.1 and 15.3 Hz, 1H); 7.05 (d, J=8.5Hz, 1H); 7.18 (dd, J=2.1 and 8.5 Hz, 1H); 7.22 (d, J=8.4 Hz, 2H); 7.25(d, J=8.4 Hz, 2H); 7.29 (d, J=2.1 Hz, 1H); 7.31 (m, 1H); 7.77 to 8.81(m, 5H). LCMS (A): ES m/z=1227 [M−H]⁻; m/z=1229 [M+H]⁺; m/z=615;t_(R)=1.16 min.

Example 31: 2,5-dioxopyrrolidin-1-yl5-(((S)-1-(((S)-1-((4-((2R,3R)-3-((S)-1-((3S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13-en-16-yl)ethyl)oxiran-2-yl)benzyl)amino)-1-oxo-6-((2S,3S,4S,5R,6R)-3,4,5-trihydroxy-6-methoxytetrahydro-2H-pyran-2-carboxamido)hexan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-5-oxopentanoate

To example 30 (14 mg, 11.4 μmol), under magnetic stirring, were addedDMF (3 mL), DSC (4.2 mg, 15.6 μmol) and DIEA (6 μL, 34.5 μmol). Thereaction medium was stirred at RT for 4 h. At this time, the medium wasdiluted with MeTHF (10 mL) and washed with H₂O (5 mL). The aqueous phasewas extracted with MeTHF (10 mL), the combined organic phases were driedover MgSO₄, filtered concentrated in vacuo and purified by flashchromatography on 4 g of silica gel (gradient elution DCM/iPrOH) to give3.5 mg of example 31 (23%).

RMN ¹H (500 MHz, δ in ppm, DMSO-d6): 0.78 (d, J=6.6 Hz, 6H); 0.82 (d,J=7.1 Hz, 3H); 0.84 (d, J=7.1 Hz, 3H); 0.99 (s, 3H); 1.04 (d, J=6.6 Hz,3H); 1.12 (s, 3H); 1.17 to 1.70 (m, 9H); 1.77 to 1.87 (m, 3H); 1.98 (m,1H); 2.23 to 2.32 (m, 3H); 2.65 to 2.70 (m, 4H); 2.81 (s, 4H); 2.93 to3.07 (m, 6H); 3.15 (m, 1H); 3.28 to 3.38 (partially masked m, 2H); 3.40(s, 3H); 3.53 (d, J=9.6 Hz, 1H); 3.81 (s, 3H); 3.87 (d, J=1.4 Hz, 1H);4.09 (d, J=7.7 Hz, 1H); 4.17 (dd, J=6.9 and 8.2 Hz, 1H); 4.20 to 4.33(m, 4H); 4.90 (dd, J=3.4 and 9.5 Hz, 1H); 5.04 (m, 2H); 5.10 (m, 1H);5.14 (d, J=5.2 Hz, 1H); 5.79 (dd, J=0.8 and 15.3 Hz, 1H); 6.47 (m, 1H);7.05 (d, J=8.8 Hz, 1H); 7.17 (dd, J=1.6 and 8.8 Hz, 1H); 7.20 to 7.25(m, 5H); 7.28 (d, J=1.6 Hz, 1H); 7.86 to 7.99 (m, 3H); 8.37 (m, 2H).LCMS (A): ES m/z=1370 [M−H+HCO₂H]⁻; ES m/z=1326 [M+H]⁺; m/z=663.5;t_(R)=1.21 min.

Example 32: hu2H11_R35-74-Ex31

The general method described previously was used for the preparation ofexample 32. 27.9 mg of hu2H11_R35-74 were reacted with 107 μL of a 10.5mM solution of example 31 in DMA (6 eq.) for 2 h 30 then were added 107μL of a 10.5 mM solution of example 31 in DMA (6 eq.) for 4 h 30. Afterpurification on Superdex 200 pg in buffer B pH 6.5+10% NMP,concentration on Amicon Ultra-15, dilution in buffer B pH 6.5 to a finalconcentration of NMP at 5% and filtration on 0.22 μm PVDF filter, 16.7mg of example 32 were obtained as a colorless limpid solution at aconcentration of 2.23 mg/mL with a DAR of 3 (HRMS), a monomeric purityof 100% and a global yield of 60%.

SEC-HRMS: m/z=149407 (naked mAb); m/z=150619 (D1); m/z=151831 (D2);m/z=153044 (D3); m/z=154256 (D4); m/z=155468 (D5); m/z=156685 (D6);m/z=157899 (D7); m/z=159014 (D8).

Synthesis of Examples 33 to 35: glutaryl-Val-sulfoPEG4Lys-C52 BenzylicAmine, NHS Ester of glutaryl-Val-sulfoPEG4Lys-C52 Benzylic Amine andCorresponding ADC

Compound 70: di-tert-butyl 4,7,10,13-tetraoxahexadecane-1,16-dioate

To a solution of triethylene glycol (5 mL, 37.5 mmol) in THF (15 mL) wasadded sodium (18 mg, 783.0 μmol). The reaction medium was stirred for 2h at RT then was added dropwise over 5 min tert-butyl acrylate (14 mL,95.58 mmol). The reaction medium was stirred for 20 h at RT thenquenched with brine (300 mL) and extracted twice with EtOAc (300 mL).The combined organic phases were dried over MgSO₄, filtered,concentrated in vacuo and purified by flash chromaography on 200 g ofsilica gel (gradient elution heptane/EtOAc) to five 8.05 g of compound70 as a colorless oil (53%).

RMN ¹H (400 MHz, δ in ppm, DMSO-d6): 1.40 (s, 18H); 2.40 (t, J=6.5 Hz,4H); 3.48 (m, 12H); 3.58 (t, J=6.5 Hz, 4H). LCMS (A): ES m/z=407 [M+H]⁺;m/z=429 [M+Na]⁺; t_(R)=1.32 min.

Compound 71: 4,7,10,13-tetraoxahexadecane-1,16-dioic Acid

To compound 70 (8.05 g, 19.8 mmmol) was added formic acid (40 mL, 1.04mol). The reaction medium was stirred for 1 d at RT, concentrated invacuo and co-evaporated with EtOAc (20 mL) to give 5.9 g of compound 71as a colorless oil (quant.).

RMN ¹H (300 MHz, δ in ppm, DMSO-d6): 2.44 (t, J=6.5 Hz, 4H); 3.48 (s,8H); 3.50 (s, 4H); 3.59 (d, J=6.5 Hz, 4H); 12.25 (broad m, 2H).

Compound 72: 3-oxo-1-phenyl-2,6,9,12,15-pentaoxaoctadecan-18-oic Acid

To a solution of compound 71 (5.9 g, 20.05 mmol) in DMF (50 mL) wereadded DIEA (3.6 mL, 20.67 mmol) and dropwise over 10 min a solution ofbenzyl bromide (2.43 mL, 20.05 mmol) in DMF (50 mL). The reaction mediumwas stirred for 20 h at RT, quenched with 1N HCl (500 mL) and extractedtwice with EtOAc (200 mL). The combined organic phases were dried overMgSO₄, filtered, concentrated in vacuo and purified by two consecutiveflash chromatographies on 400 g and 100 g of silica gel (gradientelution DCM/MeOH) to give 2.7 g of compound 72 as a pale green oil(35%).

RMN ¹H (300 MHz, δ in ppm, DMSO-d6): 2.42 (m, 2H); 2.60 (t, J=6.5 Hz,2H); 3.47 (s, 6H); 3.49 (s, 6H); 3.59 (m, 2H); 3.66 (d, J=6.5 Hz, 2H);5.10 (s, 2H); 7.29 to 7.41 (m, 5H).

Compound 73: sodium 2-aminoethanesulfonate

To a solution of taurine (2 g, 15.98 mmol) in H₂O (30 mL) was addedsodium hydrogenocarbonate (1.34 g, 15.98 mmol). The reaction medium wasstirred for 2 h at RT then concentrated in vacuo. The crude product wasdissolved in H₂O (100 mL) and lyophilized to give 2.5 g of compound 73as a white powder (quant.).

IR spectrum as a KBr pellet; main absorption bands in reciprocalcentimeters: 1458; 1344; 1212; 1184; 1046; 1037; 742; 737; 598; 576; 533& 523.

Compound 74: sodium3,18-dioxo-1-phenyl-2,6,9,12,15-pentaoxa-19-azahenicosane-21-sulfonate

To a solution of compound 72 (1 g, 2.69 mmol) in DMF (40 mL) were addedNHS (367 mg, 3.13 mmol) and EDC (565 μL, 3.12 mmol). The reaction mediumwas stirred for 20 h at RT then were added a solution of compound 73(1.9 g, 12.91 mmol) in H₂O (20 mL) and sodium bicarbonate (219 mg, 2.61mmol). The reaction medium was stirred for 2 h at RT then acidified topH 3 with Amberlite IR-120 (H) (CAS number [78922-04-0], filtered,concentrated in vacuo and purified by flash chromatography on 150 g ofsilica gel (DCM/MeOH/H₂O) to give 824 mg of compound 74 as a colorlesssolid 61%).

LCMS (A): ES m/z=490 [M−H]⁻; m/z=492 [M+H]⁺; t_(R)=1.1 min.

Compound 75: sodium1-carboxy-15-oxo-3,6,9,12-tetraoxa-16-azaoctadecane-18-sulfonate

A solution of compound 74 (820 mg, 1.60 mmol) in 8:2 MeOH/H₂O (30 mL)was filtered over 0.22 μm and subjected to hydrogenolysis on a 10% Pd/Ccartridge using a H-cube system at 20° C. The reaction medium wasconcentrated in vacuo to give 530 mg of compound 75 as a yellow gum(78%).

RMN ¹H (300 MHz, δ in ppm, DMSO-d6): 2.27 (t, J=6.6 Hz, 2H); 2.40 (t,J=6.6 Hz, 2H); 2.52 (partially masked m, 2H); 3.28 (partially masked m,2H); 3.49 (m, 12H); 3.59 (m, 4H); 7.72 (broad t, J=6.4 Hz, 1H); 12.08(broad m, 1H).

Compound 76: Sodium(5S,8S)-8-carboxy-1-(9H-fluoren-9-yl)-5-isopropyl-3,6,14,29-tetraoxo-2,17,20,23,26-pentaoxa-4,7,13,30-tetraazadotriacontane-32-sulfonate

To a solution of compound 75 (300 mg, 708.5 μmol) in DMF (15 mL) wereadded NHS (98 mg, 851.5 μmol) and EDC (150 μL, 855.1 μmol). The reactionmedium was stirred for 20 h at RT then were added a solution of compound64 (416 mg, 825.4 μmol) in DMF (15 mL) and a solution of sodiumbiacarbonate (180 mg, 2.13 mmol) in H₂O (10 mL). The reaction medium wasstirred for 1 h at RT, acidified to pH 3 with Amberlit IR-120 (H) (CASnumber [78922-04-0]), filtered, washed with DMF (5 mL), concentrated invacuo and purified by flash chromatography on 100 g of silica gel(gradient elution DCM/MeOH/acetic acid) to give 447 mg of a colorlesslacquer. This lacquer was dissolved in 2:1 THF/H₂O (120 mL), acidifiedto pH 3.3 with 0.1N HCl (about 4 mL) and concentrated in vacuo to give354 mg of compound 76 as a white translucent solid (57%).

RMN ¹H (400 MHz, δ in ppm, DMSO-d6): 0.86 (d, J=7.0 Hz, 3H); 0.89 (d,J=7.0 Hz, 3H); 1.20 to 1.41 (m, 4H); 1.50 to 1.74 (m, 2H); 1.98 (m, 1H);2.26 (t, J=6.8 Hz, 2H); 2.28 (t, J=6.8 Hz, 2H); 2.52 (masked m, 2H);3.00 (m, 2H); 3.28 (partially masked m, 2H); 3.45 (m, 12H); 3.58 (t,J=6.8 Hz, 4H); 3.91 (dd, J=7.0 and 9.2 Hz, 1H); 4.14 (m, 1H); 4.19 to4.31 (m, 3H); 7.32 (m, 2H); 7.37 (d, J=9.2 Hz, 1H); 7.41 (dt, J=1.0 and7.8 Hz, 2H); 7.75 (m, 3H); 7.80 (t, J=6.0 Hz, 1H); 7.89 (d, J=7.8 Hz,2H); 8.07 (d, J=8.0 Hz, 1H); 12.49 (m, 1H). LCMS (A): ES m/z=849 [M−H]⁻;m/z=851 [M+H]⁺; t_(R)=1.44 min.

Compound 77: sodium(25S,28S)-28-amino-25-((4-((2R,3R)-3-((S)-1-((3S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclo-hexadec-13-en-16-yl)ethyl)oxiran-2-yl)benzyl)carbamoyl)-29-methyl-4,19,27-trioxo-7,10,13,16-tetraoxa-3,20,26-triazatriacontane-1-sulfonate

To a solution(E)-(3S,10R,16S)-16-{(S)-1-[(2R,3R)-3-(4-aminomethyl-phenyl)-oxiranyl]-ethyl}-10-(3-chloro-4-methoxy-benzyl)-3-isobutyl-6,6-dimethyl-1,4-dioxa-8,11-diaza-cyclohexadec-13-ene-2,5,9,12-tetraone (thesynthesis of which was described in WO2011001052, compound 77, 106 mg,151.8 μmol) in DMF (5 mL) were added a solution of compound 76 (225 mg,257.7 μmol) in DMF (5 mL), HOBt (30 mg, 213.1 μmol) and EDC (40 μL,226.0 μmol). The reaction medium as stirred for 2 h at RT then was addedpiperidine (134 μL, 1.36 mmol) and the stirring was carried on for 2 h.The reaction medium was concentrated in vacuo and purified by twoconsecutive flash chromatographies on 50 g and 4 g of silica gel(gradient elution DCM/MeOH/H₂O) to give 73 mg of compound 77 as a whitesolid (36%).

RMN ¹H (500 MHz, δ in ppm, DMSO-d6): 0.78 (d, J=6.8 Hz, 6H); 0.91 (d,J=7.0 Hz, 3H); 0.93 (d, J=7.0 Hz, 3H); 1.00 (s, 3H); 1.04 (d, J=7.1 Hz,3H); 1.12 (s, 3H); 1.18 to 1.45 (m, 5H); 1.51 to 1.72 (m, 4H); 1.80 (m,1H); 2.05 (m, 1H); 2.23 to 2.35 (m, 5H); 2.56 (m, 2H); 2.68 (m, 2H);2.95 to 3.04 (m, 5H); 3.32 (m, 3H); 3.48 (m, 12H); 3.57 (t, J=6.7 Hz,2H); 3.59 (t, J=6.7 Hz, 2H); 3.66 (d, J=6.0 Hz, 1H); 3.80 (s, 3H); 3.87(d, J=2.3 Hz, 1H); 4.20 to 4.36 (m, 4H); 4.90 (dd, J=3.8 and 9.8 Hz,1H); 5.12 (m, 1H); 5.80 (dd, J=2.0 and 15.2 Hz, 1H); 6.47 (ddd, J=3.8,11.3 and 15.2 Hz, 1H); 7.05 (d, J=8.6 Hz, 1H); 7.16 (dd, J=2.4 and 8.6Hz, 1H); 7.24 (m, 5H); 7.29 (d, J=2.4 Hz, 1H); 7.76 (t, J=6.0 Hz, 1H);7.86 (t, J=8.0 Hz, 1H); 7.93 (broad m, 3H); 8.38 (d, J=8.0 Hz, 1H); 8.49(d, J=8.0 Hz, 1H); 8.59 (t, J=6.0 Hz, 1H). LCMS (A): ES m/z=655[M+2H]²⁺; m/z=1306 [M−H]⁻; m/z=1308 [M+H]⁺; t_(R)=1.07 min.

Example 33: Sodium(25S,28S)-33-carboxy-25-((4-((2R,3R)-3-((S)-1-((3S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclo-hexadec-13-en-16-yl)ethyl)oxiran-2-yl)benzyl)carbamoyl)-28-isopropyl-4,19,27,30-tetraoxo-7,10,13,16-tetraoxa-3,20,26,29-tetraazatritriacontane-1-sulfonate

To a solution of compound 77 (73 mg, 54.9 μmol) in DMF was addedglutaric anhydride (11 mg, 96.4 μmol). The reaction medium was stirredfor 20 h at RT, concentrated in vacuo and purified by flashchromatography on 4 g of silica gel (DCM/MeOH/H₂O) to give 40 mg ofexample 33 as a white lacquer (50%).

RMN ¹H (400 MHz, δ in ppm, DMSO-d6): 0.81 (s, 9H); 1.04 (d, J=7.0 Hz,3H); 1.38 (s, 9H); 1.50 (dd, J=3.0 and 14.5 Hz, 1H); 1.58 (dd, J=9.1 and14.5 Hz, 1H); 2.38 to 2.48 (partially masked m, 3H); 4.04 (m, 1H); 4.19(m, 2H); 4.30 (m, 1H); 4.49 (s, 2H); 4.91 (m, 1H); 5.80 (d, J=16.0 Hz,1H); 6.17 (dd, J=8.4 and 16.0 Hz, 1H); 6.43 (d, J=16.0 Hz, 1H); 6.70 (m,1H); 7.23 to 7.44 (m, 8H); 7.68 (m, 2H); 7.79 (d, J=8.3 Hz, 1H); 7.88(d, J=7.8 Hz, 2H). LCMS (A): ES m/z=712 [M+2H]²⁺; m/z=1420 [M−H]⁻;m/z=1422 [M+H]⁺; t_(R)=1.57 min.

Example 34: Sodium(25S,28S)-25-((4-((2R,3R)-3-((S)-1-((3S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13-en-16-yl)ethyl)oxiran-2-yl)benzyl)carbamoyl)-34-((2,5-dioxopyrrolidin-1-yl)oxy)-28-isopropyl-4,19,27,30,34-pentaoxo-7,10,13,16-tetraoxa-3,20,26,29-tetraazatetratriacontane-1-sulfonate

To a solution of example 33 (3.57 mg, 2.47 μmol) in DMA (93 μL) wereadded NHS (27.2 μL, 2.72 μmol) and EDC (111 μL, 2.22 μmol). The reactionmedium was stirred for 48 h at RT: LCMS analysis showed that thepresence of expected example 34 at 95% with 5% of residual example 33.This solution was used such as for conjugation and the preparation ofexample 35.

Example 35: hu2H11_R35-74-Ex34

The general method described previously was used for the preparation ofexample 35. 11.16 mg of hu2H11_R35-74 were reacted with 53 μL of a 10 mMsolution of example 34 in DMA (7 eq.) for 2 h 30 then were added 15 μLof a 10 mM solution of example 34 in DMA (2 eq.) for 2 h 30. Afterpurification on PD-10 and Nap-10 columns in buffer B pH 6.5+5% NMP, 9.5mg of example 34 were obtained as a colorless limpid solution at aconcentration of 1.9 mg/mL with a DAR of 3.8 (HRMS), a monomeric purityof 97.5% and a global yield of 82%.

SEC-HRMS: m/z=149330 (naked mAb); m/z=150744 (D1); m/z=152147 (D2);m/z=153554 (D3); m/z=154959 (D4); m/z=156366 (D5); m/z=157771 (D6);m/z=159172 (D7); m/z=160578 (D8).

Synthesis of Examples 36 & 37: glutaryl-Val-PEG4Lys-PABA-C52 BenzylicAmine and NHS Ester of glutaryl-Val-PEG4Lys-PABA-C52 Benzylic Amine

Compound 78: (9H-fluoren-9-yl)methyl((20S,23S)-20-((4-(hydroxymethyl)phenyl)carbamoyl)-24-methyl-14,22-dioxo-2,5,8,11-tetraoxa-15,21-diazapentacosan-23-yl)carbamate

To a solution of compound 61 (180 mg, 257.2 μmol) in MeCN (35 mL) wereadded, under Ar, DIEA (130 μL, 771.7 μmol), 4-aminobenzyl alcohol (48mg, 385.8 μmol), HOAt (41 mg, 295.8 μmol), HATU (147 mg, 385.8 μmol).The reaction medium was stirred for 17 h at RT then were added MeOH (30mL) and silica, the mixture was concentrated in vacuo and purified byflash chromatography on 30 g of silica gel (gradient elution DCM/MeOH)to give 94 mg of compound 78 as a beige solid (46%).

RMN ¹H (300 MHz, δ in ppm, DMSO-d6): 0.85 (d, J=7.0 Hz, 3H); 0.88 (d,J=7.0 Hz, 3H); 1.18 to 1.45 (m, 4H); 1.52 to 1.70 (m, 2H); 1.99 (m, 1H);2.27 (m, 2H); 3.00 (m, 2H); 3.22 (s, 3H); 3.35 to 3.51 (m, 12H); 3.55(t, J=6.8 Hz, 2H); 3.90 (dd, J=7.2 and 9.1 Hz, 1H); 4.18 to 4.56 (m,4H); 4.42 (d, J=5.9 Hz, 2H); 5.07 (t, J=5.9 Hz, 1H); 7.22 (d, J=8.9 Hz,2H); 7.31 (dt, J=1.8 and 7.8 Hz, 2H); 7.40 (m, 3H); 7.53 (d, J=8.9 Hz,2H); 7.73 (m, 3H); 7.88 (d, J=7.8 Hz, 2H); 8.02 (d, J=8.2 Hz, 1H); 9.92(s, 1H).

Compound 79: (9H-fluoren-9-yl)methyl((20S,23S)-24-methyl-20-((4-((((4-nitrophenoxy)-carbonyl)oxy)methyl)phenyl)carbamoyl)-4,22-dioxo-2,5,8,11-tetraoxa-15,21-diazapentacosan-23-yl)carbamate

To a solution of compound 78 (94 mg, 118.9 μmol) in DMF were added bis(4-nitrophenyl) carbonate (112 mg, 357.1 μmol) and DIEA (40 μL, 237.2μmol). The reaction medium was stirred 20 h at RT, concentrated invacuo, diluted with DCM (10 mL) and washed with H₂O (10 mL). The organicphase was dried over MgSO₄, filtered, concentrated in vacuo and purifiedby flash chromatography on 12 g of silica gel (gradient elutionDCM/MeOH) to give 55 mg of compound 79 as a brown solid (48%).

RMN ¹H (400 MHz, δ in ppm, DMSO-d6): 0.86 (d, J=7.0 Hz, 3H); 0.88 (d,J=7.0 Hz, 3H); 1.18 to 1.45 (m, 4H); 1.55 to 1.77 (m, 2H); 1.99 (m, 1H);2.27 (t, J=6.8 Hz, 2H); 3.00 (m, 2H); 3.21 (s, 3H); 3.40 to 3.52 (m,12H); 3.57 (t, J=6.8 Hz, 2H); 3.91 (m, 1H); 4.20 to 4.41 (m, 4H); 5.24(s, 2H); 7.31 (t, J=7.8 Hz, 2H); 7.35 to 7.48 (m, 5H); 7.58 (d, J=9.2Hz, 2H); 7.63 (d, J=8.8 Hz, 2H); 7.74 (m, 2H); 7.80 (t, J=6.0 Hz, 1H);7.90 (d, J=7.8 Hz, 2H); 8.10 (d, J=8.0 Hz, 1H); 8.32 (d, J=9.2 Hz, 2H);10.12 (s, 1H). LCMS (A): ES m/z=956 [M+H]⁺; m/z=978 [M+Na]⁺; m/z=1000[M−H+HCO₂H]⁻; t_(R)=1.45 min.

Compound 80:4-((S)-20-((S)-2-amino-3-methylbutanamido)-14-oxo-2,5,8,11-tetraoxa-15-azahenicosanamido)benzyl4-((2R,3R)-3-((S)-1-((3S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13-en-16yl)ethyl)-oxiran-2-yl)benzylcarbamate

To a solution(E)-(3S,10R,16S)-16-{(S)-1-[(2R,3R)-3-(4-aminomethyl-phenyl)-oxiranyl]-ethyl}-10-(3-chloro-4-methoxy-benzyl)-3-isobutyl-6,6-dimethyl-1,4-dioxa-8,11-diaza-cyclohexadec-13-ene-2,5,9,12-tetraone (thesynthesis of which was described in WO2011001052, compound 77, 19 mg,27.2 μmol) in DCM (2.5 mL) were added a solution of compound 79 (58 mg,60.7 μmol) in DCM (2.5 mL) and DIEA (15 μL, 86.6 μmol). The reactionmedium was stirred for 20 h at RT, concentrated in vacuo and purified byflash chromatography on 4 g of silica gel (gradient elution DCM/MeOH) togive 38 mg of Fmoc-protected intermediate as a colorless lacquer (92%).

RMN ¹H (400 MHz, δ in ppm, DMSO-d6): 0.79 (d, J=6.7 Hz, 6H); 0.86 (d,J=7.0 Hz, 3H); 0.88 (d, J=7.0 Hz, 3H); 1.00 (s, 3H); 1.03 (d, J=7.0 Hz,3H); 1.12 (s, 3H); 1.20 to 1.45 (m, 5H); 1.50 to 1.75 (m, 4H); 1.80 (m,1H); 2.00 (m, 1H); 2.27 (m, 3H); 2.69 (m, 2H); 2.92 to 3.04 (m, 5H);3.21 (s, 3H); 3.32 (masked m, 1H); 3.35 to 3.51 (m, 12H); 3.56 (t, J=6.6Hz, 2H); 3.80 (s, 3H); 3.87 (d, J=2.2 Hz, 1H); 3.91 (dd, J=7.1 and 9.1Hz, 1H); 4.15 to 4.42 (m, 7H); 4.90 (dd, J=3.7 and 9.8 Hz, 1H); 4.97 (s,2H); 5.10 (m, 1H); 5.79 (dd, J=2.0 and 15.2 Hz, 1H); 6.48 (ddd, J=3.8,11.1 and 15.2 Hz, 1H); 7.04 (d, J=8.6 Hz, 1H); 7.17 (dd, J=2.4 and 8.6Hz, 1H); 7.20 to 7.34 (m, 10H); 7.41 (m, 3H); 7.58 (d, J=8.6 Hz, 2H);7.70 to 7.80 (m, 4H); 7.89 (d, J=7.8 Hz, 2H); 8.06 (d, J=7.6 Hz, 1H);8.35 (d, J=8.3 Hz, 1H); 10.03 (s, 1H). LCMS (A): ES m/z=698; m/z=1514[M+H]⁺; m/z=1558 [M−H+HCO₂H]⁻; t_(R)=1.56 min.

To a solution of this intermediate (35 mg, 23.1 μmol) in DCM (5 mL) wasadded piperidine (20 μL, 201.5 μmol). The reaction medium was stirredfor 1 h 30 at RT then DMF (1 mL) was added and stirring carried on for 1h 30. Then was added piperidine (2 μL); the reaction medium was stirredfor 1 h at RT, concentrated in vacuo and purified by flashchromatography on 4 g of silica gel (gradient elution DCM/MeOH) to give25 mg of compound 80 as a colorless lacquer (83%).

RMN ¹H (400 MHz, δ in ppm, DMSO-d6): 0.78 (d, J=6.7 Hz, 6H); 0.82 (d,J=7.0 Hz, 3H); 0.89 (d, J=7.0 Hz, 3H); 1.00 (s, 3H); 1.04 (d, J=7.1 Hz,3H); 1.11 (s, 3H); 1.22 to 1.48 (m, 5H); 1.51 to 1.75 (m, 6H); 1.80 (m,1H); 1.98 (m, 1H); 2.28 (m, 3H); 2.69 (m, 2H); 2.92 to 3.05 (m, 7H);3.15 (m, 1H); 3.21 (s, 3H); 3.32 (masked m, 1H); 3.35 to 3.51 (m, 12H);3.57 (t, J=6.7 Hz, 2H); 3.80 (s, 3H); 3.87 (d, J=2.2 Hz, 1H); 4.19 (d,J=6.4 Hz, 2H); 4.23 (ddd, J=3.9, 8.3 and 11.8 Hz, 1H); 4.43 (m, 2H);4.90 (dd, J=3.7 and 9.8 Hz, 1H); 4.98 (s, 2H); 5.10 (m, 1H); 5.80 (dd,J=2.0 and 15.2 Hz, 1H); 6.48 (ddd, J=3.9, 11.3 and 15.2 Hz, 1H); 7.05(d, J=8.6 Hz, 1H); 7.18 (dd, J=2.4 and 8.6 Hz, 1H); 7.20 to 7.32 (m,8H); 7.59 (d, J=8.6 Hz, 2H); 7.79 (t, J=6.0 Hz, 1H); 7.82 (t, J=6.4 Hz,1H); 8.21 (m, 1H); 8.39 (d, J=8.3 Hz, 1H); 10.13 (s, 1H). LCMS (A): ESm/z=646.5; m/z=1292 [M+H]⁺; t_(R)=1.05 min.

Example 36:(20S,23S)-20-((4-((((4-((2R,3R)-3-((S)-1-((3S,10R,16S,E)-10-(3-chloro-4-methoxy-benzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13-en-16-yl)ethyl)oxiran-2-yl)benzyl)carbamoyl)oxy)methyl)-phenyl)carbamoyl)-23-isopropyl-14,22,25-trioxo-2,5,8,11-tetraoxa-15,21,24-triazanonacosan-29-oicAcid

To a solution of compound 80 (25 mg, 19.3 μmol) in DCM (5 mL) was addedglutaric anhydride (4 mg, 19.3 μmol). The reaction medium was stirredfor 2 h 30 at RT then were added glutaric anhydride (4 mg, 19.3 μmol)and DMF (0.5 mL) and stirring carried on for 26 h. The reaction mediumwas concentrated in vacuo and purified by flash chromatography on 4 g ofsilica gel (gradient elution DCM/MeOH) to give 10 mg of example 36 as awhite lacquer (37%).

Example 37: (20S,23S)-2,5-dioxopyrrolidin-1-yl20-((4-((((4-((2R,3R)-3-((S)-1-((3S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13-en-16-yl)ethyl)oxiran-2-yl)benzyl)-carbamoyl)oxy)methyl)phenyl)-carbamoyl)-23-isopropyl-14,22,25-trioxo-2,5,8,11-tetraoxa-15,21,24-triazanonacosan-29-oate

To a solution of example 36 (10 mg, 7.11 μmol) in THF (2 mL) were addedDSC (2 mg, 7.65 μmol) and DIEA (1.2 μL, 7.26 μmol). The reaction mediumwas stirred for 20 h at RT, concentrated in vacuo and purified by twoconsecutive flash chromatographies on 0.6 g and 2 g of silica gel(gradient elution DCM/iPrOH) to give 3.3 mg of example 37 as a whitelacquer (31%).

Synthesis of Examples 38 & 39: glutaryl-Val-PEG24Lys-PABA-C52 BenzylicAmine and NHS Ester of glutaryl-Val-PEG24Lys-PABA-C52 Benzylic Amine

Compound 81: (9H-fluoren-9-yl)methyl((80S,83S)-80-((4-(hydroxymethyl)phenyl)carbamoyl)-84-methyl-74,82-dioxo2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71-tetracosaoxa-75,81-diazapentaoctacontan-83-yl)carbamate

To a solution of compound 65 (360 mg, 229.8 μmol) in MeCN (70 mL) wereadded DIEA (77 μL, 456.6 μmol), 4-aminobenzyl alcohol (43 mg, 342.2μmol), HOAt (36 mg, 264.5 μmol) and HATU (109 mg, 278.1 μmol). Thereaction medium was stirred for 2 h at RT, concentrated in vacuo andpurified by flash chromatography on 25 g of silica gel (gradient elutionDCM/MeOH/H₂O) to give 307 mg of compound 81 as a white solid (80%).

RMN ¹H (400 MHz, δ in ppm, DMSO-d6): 0.85 (d, J=7.0 Hz, 3H); 0.88 (d,J=7.0 Hz, 3H); 1.21 to 1.42 (m, 4H); 1.54 to 1.78 (m, 2H); 1.99 (m, 1H);2.27 (t, J=6.8 Hz, 2H); 3.00 (m, 2H); 3.22 (s, 3H); 3.40 to 3.52 (m,92H); 3.54 (t, J=6.8 Hz, 2H); 3.91 (dd, J=6.9 and 9.0 Hz, 1H); 4.18 to4.32 (m, 3H); 4.39 (m, 1H); 4.42 (d, J=5.9 Hz, 2H); 5.08 (t, J=5.9 Hz,1H); 7.22 (d, J=8.9 Hz, 2H); 7.32 (dt, J=1.2 and 7.8 Hz, 2H); 7.41 (m,3H); 7.53 (d, J=8.9 Hz, 2H); 7.75 (m, 3H); 7.89 (d, J=7.8 Hz, 2H); 8.02(d, J=8.2 Hz, 1H); 9.92 (s, 1H). LCMS (d): ES m/z=827; m/z=1671 [M+H]⁺;t_(R)=2.94 min.

Compound 82: (9H-fluoren-9-yl)methyl((80S,83S)-84-methyl-80-((4-((((4-nitrophenoxy)-carbonyl)oxy)methyl)phenyl)carbamoyl)-74,82-dioxo-2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71-tetracosaoxa-75,81-diazapentaoctacontan-83-yl)carbamate

To a solution of compound 81 (300 mg, 179.4 μmol) in DCM (10 mL) wereadded bis (4-nitrophenyl) carbonate (450 mg, 1.48 mmol) and DIEA (60 μL,361.2 μmol). The reaction medium was stirred at RT overnight then wasadded bis (4-nitrophenyl) carbonate (80 mg, 263.1 μmol) and stirringcarried on for 4 h. At that time was added bis (4-nitrophenyl) carbonate(80 mg, 263.1 μmol) and stirring carried on overnight. The reactionmedium was concentrated in vacuo and purified by flash chromatography on80 g of silica gel (gradient elution DCM/MeOH/H₂O) to give 325 mg ofcompound 82 as a white lacquer (98%).

RMN ¹H (400 MHz, δ in ppm, DMSO-d6): 0.86 (d, J=7.0 Hz, 3H); 0.89 (d,J=7.0 Hz, 3H); 1.20 to 1.45 (m, 4H); 1.58 to 1.76 (m, 2H); 1.99 (m, 1H);2.27 (t, J=6.8 Hz, 2H); 3.00 (m, 2H); 3.22 (s, 3H); 3.40 to 3.70 (m,94H); 3.91 (dd, J=7.2 and 9.1 Hz, 1H); 4.19 to 4.32 (m, 3H); 4.39 (m,1H); 5.24 (s, 2H); 7.31 (dt, J=1.2 and 7.8 Hz, 2H); 7.40 (m, 4H); 7.57(d, J=9.2 Hz, 2H); 7.62 (d, J=8.6 Hz, 2H); 7.73 (m, 3H); 7.89 (d, J=7.8Hz, 2H); 8.07 (d, J=7.8 Hz, 1H); 8.31 (d, J=9.2 Hz, 2H); 10.10 (s, 1H).LCMS (d): ES m/z=827; m/z=1836 [M+H]⁺; t_(R)=3.38 min.

Compound 83:4-((S)-80-((S)-2-amino-3-methylbutanamido)-74-oxo-2,5,8,11,14,17,20,23,26,29,32,35,38,41,47,50,53,56,59,62,65,68,71-tetracosaoxa-75-azahenoctacontanamido)-benzyl4-((2R,3R)-3-((S)-1-((3S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13-en-16-yl)ethyl)oxiran-2-yl)benzyl-carbamate

To a solution of(E)-(3S,10R,16S)-16-{(S)-1-[(2R,3R)-3-(4-aminomethyl-phenyl)-oxiranyl]-ethyl}-10-(3-chloro-4-methoxy-benzyl)-3-isobutyl-6,6-dimethyl-1,4-dioxa-8,11-diaza-cyclohexadec-13-ene-2,5,9,12-tetraone (thesynthesis of which was described in WO2011001052, compound 77, 79 mg,113.1 μmol) in DCM (5 mL) were added a solution of compound 82 (325 mg,177.5 μmol) in DCM (5 mL) and DIEA (63 μL, 365.6 μmol). The reactionmedium was stirred for 20 h at RT then was added compound 82 (34 mg,18.6 μmol) and stirring carried on for 4 h. The reaction medium wasconcentrated in vacuo and purified by two consecutive flashchromatographies on 25 g of silica gel and 15 g of diol-modified silicagel (gradient elution DCM/MeOH) to give 152 mg of crude product that waspurified by reverse phase chromatography on a 10 μm C18 column 250×40 mm(gradient elution MeCN/H₂O) to give 78 mg of the Fmoc-protectedintermediate containing 16% of compound 83 as a white powder (29%).

RMN ¹H (500 MHz, δ in ppm, DMSO-d6): 0.78 (d, J=6.8 Hz, 6H); 0.85 (d,J=7.0 Hz, 3H); 0.88 (d, J=7.0 Hz, 3H); 1.00 (s, 3H); 1.04 (d, J=7.1 Hz,3H); 1.12 (s, 3H); 1.20 to 1.43 (m, 5H); 1.50 to 1.74 (m, 4H); 1.79 (m,1H); 1.99 (m, 1H); 2.25 (m, 3H); 2.68 (m, 2H); 2.93 to 3.04 (m, 5H);3.22 (s, 3H); 3.32 (masked m, 2H); 3.40 to 3.76 (m, 94H); 3.80 (s, 3H);3.87 (d, J=2.0 Hz, 1H); 3.90 (dd, J=7.5 and 9.1 Hz, 1H); 4.19 (d, J=6.5Hz, 2H); 4.20 to 4.33 (m, 4H); 4.39 (m, 1H); 4.90 (dd, J=3.9 and 9.9 Hz,1H); 4.93 (s, 2H); 5.11 (m, 1H); 5.79 (dd, J=1.8 and 15.2 Hz, 1H); 6.47(ddd, J=3.8, 11.4 et 15.2 Hz, 1H); 7.05 (d, J=8.6 Hz, 1H); 7.17 (dd,J=2.4 and 8.6 Hz, 1H); 7.20 to 7.36 (m, 10H); 7.41 (m, 3H); 7.48 (d,J=8.6 Hz, 2H); 7.70 to 7.80 (m, 5H); 7.89 (d, J=7.8 Hz, 2H); 8.08 (d,J=8.0 Hz, 1H); 8.35 (d, J=8.0 Hz, 1H); 10.04 (s, 1H).

To a solution of this intermediate (75 mg, 31.3 μmol) in DMF (5 mL) wasadded piperidine (25 μL, 253.2 μmol). The reaction medium was stirredfor 1 h 30 at RT, concentrated in vacuo and purified by flashchromatography on 4 g of silica gel (gradient elution DCM/MeOH/H₂O) togive 58 mg of compound 83 as a white lacquer (85%).

RMN ¹H (500 MHz, δ in ppm, DMSO-d6): 0.78 (d, J=6.9 Hz, 9H); 0.88 (d,J=7.0 Hz, 3H); 1.00 (s, 3H); 1.05 (d, J=7.1 Hz, 3H); 1.12 (s, 3H); 1.15to 1.50 (m, 7H); 1.52 to 1.73 (m, 4H); 1.80 (m, 1H); 1.93 (m, 1H); 2.27(m, 3H); 2.69 (m, 2H); 2.95 to 3.04 (m, 6H); 3.23 (s, 3H); 3.32 (m, 1H);3.40 to 3.55 (m, 92H); 3.57 (t, J=6.8 Hz, 2H); 3.80 (s, 3H); 3.87 (d,J=2.2 Hz, 1H); 4.20 (d, J=6.5 Hz, 2H); 4.24 (ddd, J=3.9, 8.2 and 12.0Hz, 1H); 4.42 (m, 1H); 4.90 (dd, J=3.8 and 9.8 Hz, 1H); 4.98 (s, 2H);5.10 (m, 1H); 5.79 (d, J=15.8 Hz, 1H); 6.47 (ddd, J=3.8, 6.0 and 11.5Hz, 1H); 7.05 (d, J=8.6 Hz, 1H); 7.16 (dd, J=2.4 and 8.6 Hz, 1H); 7.20to 7.31 (m, 8H); 7.58 (d, J=8.6 Hz, 2H); 7.78 (m, 2H); 8.10 (d, J=8.6Hz, 1H); 8.36 (d, J=8.1 Hz, 1H); 10.10 (s, 1H). LCMS (d): ES m/z=725;m/z=1087 [M+2H]²⁺; t_(R)=1.07 min.

Example 38:(80S,83S)-80-((4-((((4-((2R,3R)-3-((S)-1-((3S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13-en-16-yl)ethyl)oxiran-2-yl)benzyl)carbamoyl)oxy)methyl)-phenyl)carbamoyl)-83-isopropyl-74,82,85-trioxo-2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71-tetracosaoxa-75,81,84-triazanonaoctacontan-89-oicAcid

To a solution of compound 83 (58 mg, 26.7 μmol) in DCM (2 mL) was addedglutaric anhydride (5.5 mg, 48.2 μmol). The reaction medium was stirredfor 3 h at RT, concentrated in vacuo and purified by two consecutiveflash chromatographies on 4 g and 12 g of silica gel (gradient elutionDCM/MeOH/H₂O) to give 37 mg of example 38 as a white lacquer (61%).

RMN ¹H (500 MHz, δ in ppm, DMSO-d6): 0.79 (d, J=6.8 Hz, 6H); 0.93 (d,J=7.0 Hz, 3H); 0.97 (d, J=7.0 Hz, 3H); 1.00 (s, 3H); 1.04 (d, J=7.1 Hz,3H); 1.12 (s, 3H); 1.20 to 1.45 (m, 5H); 1.51 to 1.75 (m, 6H); 1.80 (m,1H); 2.00 (m, 1H); 2.13 to 2.33 (m, 7H); 2.69 (m, 2H); 2.95 to 3.05 (m,5H); 3.23 (s, 3H); 3.32 (m, 1H); 3.40 to 3.58 (m, 94H); 3.80 (s, 3H);3.87 (d, J=2.2 Hz, 1H); 4.15 to 4.38 (m, 4H); 4.32 (m, 1H); 4.90 (dd,J=3.8 and 9.8 Hz, 1H); 4.98 (s, 2H); 5.10 (m, 1H); 5.79 (d, J=15.2 Hz,1H); 6.47 (ddd, J=3.8, 11.4 and 15.2 Hz, 1H); 7.05 (d, J=8.6 Hz, 1H);7.16 (dd, J=2.4 and 8.6 Hz, 1H); 7.20 to 7.32 (m, 8H); 7.60 (d, J=8.6Hz, 2H); 7.78 (t, J=6.5 Hz, 1H); 7.90 (broad d, J=8.6 Hz, 2H); 8.20 (m,1H); 8.38 (d, J=8.6 Hz, 2H); 10.08 (m, 1H); 12.10 (m, 1H). LCMS (d): ESm/z=698; m/z=2287 [M+H]⁺; t_(R)=3.13 min.

Example 39: (80S,83S)-2,5-dioxopyrrolidin-1-yl80-((4-((((4-((2R,3R)-3-((S)-1-((3S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diaza-cyclohexadec-13-en-16-yl)ethyl)oxiran-2-yl)benzyl)carbamoyl)oxy)methyl)phenyl)carbamoyl)-83-isopropyl-74,82,85-trioxo2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71-tetracosaoxa-75,81,84-triazanonaoctacontan-89-oate

To a solution of example 38 (29 mg, 12.7 μmol) in THF (3 mL) were addedDSC (3.6 mg, 13.8 μmol) and DIEA (2.1 μL, 12.9 μmol). The reactionmedium was stirred for 20 h at RT, concentrated in vacuo and purified byflash chromatography on 4 g of silica gel (gradient elution DCM/iPrOH)to give 6.9 mg of example 39 as a colorless lacquer (23%).

RMN ¹H (500 MHz, δ in ppm, DMSO-d6): 0.79 (d, J=6.8 Hz, 6H); 0.93 (d,J=7.0 Hz, 3H); 0.97 (d, J=7.0 Hz, 3H); 1.00 (s, 3H); 1.04 (d, J=7.1 Hz,3H); 1.11 (s, 3H); 1.13 to 1.42 (m, 5H); 1.52 to 1.88 (m, 7H); 1.98 (m,1H); 2.28 (m, 5H); 2.53 to 2.72 (m, 4H); 2.80 (s, 4H); 2.96 to 3.05 (m,5H); 3.23 (s, 3H); 3.32 (m, 1H); 3.60 to 3.65 (m, 94H); 3.80 (s, 3H);3.87 (d, J=2.2 Hz, 1H); 4.15 to 4.28 (m, 4H); 4.33 (m, 1H); 4.90 (dd,J=3.8 and 9.8 Hz, 1H); 4.98 (s, 2H); 5.10 (m, 1H); 5.79 (d, J=15.8 Hz,1H); 6.47 (ddd, J=3.8, 11.4 and 15.2 Hz, 1H); 7.05 (d, J=8.6 Hz, 1H);7.17 (dd, J=2.4 and 8.6 Hz, 1H); 7.20 to 7.32 (m, 8H); 7.60 (d, J=8.6Hz, 2H); 7.78 (m, 2H); 7.90 (d, J=8.5 Hz, 1H); 8.07 (m, 2H); 8.37 (d,J=8.5 Hz, 2H); 9.95 (s, 1H). LCMS (D): ES m/z=698; m/z=2384 [M+H]⁺;t_(R)=3.19 min.

Synthesis of Examples 40 & 41: DBCO-glutaryl-Val-PEG24Lys-PABA-C52Benzylic Amine and Corresponding ADC

Compound 84:5-[[3-(11,12-didehydrodibenz[b,f]azocin-5(6H)-yl)-3-oxopropyl]amino]-5-oxopentanoicAcid

To a solution of DBCO-amine (50 mg, 180.9 μmol) in DCM (2 mL) was addedglutaric anhydride (21.1 mg, 180.9 μmol). The reaction medium wasstirred for 2 h at RT, concentrated in vacuo and purified by flashchromatography on 4 g of silica gel (gradient elution DCM/MeOH) to give32.6 mg of compound 84 (45%).

RMN ¹H (400 MHz, δ in ppm, DMSO-d6): 1.59 (m, 2H); 1.81 (m, 1H); 1.94(t, J=7.4 Hz, 2H); 2.11 (t, J=7.4 Hz, 2H); 2.40 (m, 1H); 2.91 (m, 1H);3.09 (m, 1H); 3.62 (d, J=14.2 Hz, 1H); 5.13 (d, J=14.2 Hz, 1H); 7.38 to7.53 (m, 6H); 7.56 to 7.67 (m, 3H); 12.05 (broad m, 1H).

Example 40: DBCO-glutaryl-Val-PEG24Lys-PABA-C52 Benzylic Amine

To a solution of compound 83 (22 mg, 10.1 μmol) in DCM (2 mL) were addedcompound 84 (5.14 mg, 13.2 μmol), EDC (1.8 μL, 10.1 μmol) and HOBt (1.4mg, 10.1 μmol). The reaction medium was stirred at RT overnight,concentrated in vacuo and purified by reverse phase chromatography on aC18-modified 5 μm column 150 mm×30 mm (gradient elution MeCN/0.2Nammonium acetate pH 5.6) to give 9.2 mg of example 40 as a white powder(36%).

RMN ¹H (500 MHz, δ in ppm, DMSO-d6): 0.78 (d, J=6.8 Hz, 6H); 0.80 to0.90 (m, 6H); 1.00 (s, 3H); 1.05 (d, J=7.1 Hz, 3H); 1.12 (s, 3H); 1.20to 1.41 (m, 5H); 1.52 to 1.72 (m, 6H); 1.75 to 2.00 (m, 5H); 2.09 (m,2H); 2.27 (m, 3H); 2.40 (m, 1H); 2.68 (m, 2H); 2.88 to 3.12 (m, 7H);3.23 (s, 3H); 3.28 to 3.58 (partially masked m, 95H); 3.60 (d, J=11.0Hz, 1H); 3.80 (s, 3H); 3.88 (d, J=2.0 Hz, 1H); 4.11 to 4.28 (m, 4H);4.33 (m, 1H); 4.90 (dd, J=3.8 and 9.8 Hz, 1H); 4.97 (s, 2H); 5.04 (d,J=11.0 Hz, 1H); 5.11 (m, 1H); 5.80 (d, J=15.2 Hz, 1H); 6.47 (ddd, J=3.8,11.4 and 15.2 Hz, 1H); 7.05 (d, J=8.6 Hz, 1H); 7.17 (dd, J=2.4 and 8.6Hz, 1H); 7.20 to 7.31 (m, 6H); 7.32 to 7.50 (m, 6H); 7.53 to 7.70 (m,4H); 7.80 (m, 3H); 8.08 (d, J=8.9 Hz, 1H); 8.36 (d, J=8.4 Hz, 1H); 9.95(s, 1H). LCMS (D): ES m/z=849 [M+3H]³⁺; m/z=1273 [M+2H]²⁺; t_(R)=5.29min.

Example 41: hu2H11_R35-74-Ex40

194 mg of hu2H11_R35-74 in buffer A were reacted with 627 μL of asolution of 2,5-dioxopyrrolidin-1-yl 4-azidobutanoate (CAS number[943858-70-6]) at 22.0 mM in DMA (10 eq.) such as the final antibodyconcentration was 10.5 mg/mL and the percentage of DMA in buffer A was5%. After stirring for 2 h at RT, the mixture was purified by gelfiltration using a a Sephadex™ G25 matrix (Hiprep 26/10 desaltingcolumn, GEHealthcare) pre-equilibrated in buffer A. The fractionscontaining the monomeric modified antibody were pooled and filteredthrough a Steriflip® filter unit (0.22 μm Durapore® PVDF membrane,Millipore) to provide 173 mg of modified antibody at a concentration of5.75 mg/mL with a ratio of linker per antibody of 5.2 (HRMS), amonomeric purity of 97% and a global yield of 89%.

The general method described previously was used for the preparation ofexample 41. 45 mg of modified hu2H11_R35-74 were reacted with 604 μL ofa 6.89 mM solution of example 40 in DMA (14 eq.) for 4 h 30 then wereadded 604 μL of a 10 mM solution of example 40 in DMA (14 eq.) andstirring was pursued overnight at RT. After purification on Superdex 200pg in buffer B pH 6.5+20% NMP, concentration on Amicon Ultra-15, gelfiltration on PD-10 in buffer B pH 6.5+5% NMP and filtration on 0.22 μmPVDF filter, 28 mg of example 41 were obtained as a colorless limpidsolution at a concentration of 1.6 mg/mL with a DAR of 3.44 (HRMS), amonomeric purity of 99.6% and a yield of 62%.

SEC-HRMS: m/z=152041 (D1); m/z=154678 (D2); m/z=157332 (D3); m/z=159992(D4); m/z=162648 (D5); m/z=165303 (D6); m/z=168004 (D7).

Synthesis of Example 42: sulfo-Val-PEG4Lys-C52 Benzylic Amine

Compound 85: Sodium2-(4-((3-(allyloxy)-3-oxopropyl)(3-(tert-butoxy)-3-oxopropyl)amino)-4-oxobutanamido)ethane-1-sulfonate

To a solution of compound 13 (0.958 g, 2.68 mmol) in DMF (5 mL) wereadded NHS (390.3 mg, 3.32 mmol) and EDC (604.2 μL, 3.4 mmol). Thereaction medium was stirred for 2 h at RT then stored 2 d at −20° C.After getting back at RT, the reaction medium was stirred for 2 h at RTthen were added EDC (60 μL, 340 μmol) and a solution of NHS (20 mg,170.3 μmol) in DMF (3 mL). The reaction medium was stirred at RTovernight then were added a solution of compound 73 (1.96 g, 13.3 mmol)in H₂O (10 mL) and sodium hydrogenocarbonate (225.2 mg, 2.68 mmol). Thereaction medium was stirred for 1 h at RT, diluted with H₂O (6 mL),acidified to pH 3 with Amberlite IR-120 (H) (CAS number [78922-04-4]),filtered, concentrated in vacuo and purified by flash chromatography onsilica gel (gradient elution DCM/MeOH/H₂O) to give 270 mg of compound 85as a colorless foam (21%).

RMN ¹H (400 MHz, δ in ppm, DMSO-d6): 50:50 conformer mixture; 1.39 (s,4.5H); 1.40 (s, 4.5H); 2.27 (t, J=7.0 Hz, 4H); 2.38 (t, J=7.0 Hz, 2H);3.53 (m, 3H); 2.68 (t, J=7.0 Hz, 1H); 3.25 to 3.61 (m, 6H); 4.55 to 4.58(m, 2H); 5.18 to 5.36 (m, 2H); 5.90 (m, 1H); 7.70 (t, J=6.8 Hz, 1H);10.53 (broad m, 1H). LCMS (A): ES m/z=409; m/z=463 [M−H]⁻; m/z=465[M+H]⁺; t_(R)=1.13 min.

Compound 86: sodium2-(4-((3-(allyloxy)-3-oxopropyl)(2-carboxyethyl)amino)-4-oxobutanamido)-ethane-1-sulfonate

To a solution of compound 85 (60 mg, 123.3 μmol) in DCM (4 mL) was addedTFA (100 μL, 1.25 mmol). The reaction medium was stirred for 2 h at RTthen was added TFA (100 μL, 1.25 mmol) and stirring was carried on for 2d at RT. Then was added TFA (50 μL, 0.625 mmol), the reaction medium wasstirred at RT, concentrated in vacuo and co-evaporated with toluene (3×)to give 60 mg of compound 86 as a colorless lacquer (quant.).

Compound 87: Sodium2-(4-((3-(allyloxy)-3-oxopropyl)(3-((2,5-dioxopyrrolidin-1-yl)oxy)-3-oxopropyl)amino)-4-oxobutanamido)ethane-1-sulfonate

To a solution of compound 86 (52.94 mg, 123 μmol) in DCM (5 mL) wereadded EDAC (26.0 μL, 147.6 μmol), NHS (14.75 mg, 123 μmol) and DMF (0.5mL). The reaction mixture was stirred for 4 h at RT then was added EDC(10 μL, 45.7 μmol) and stirring was carried on at RT overnight. Thereaction medium was stored at −20° C. and used such as for the synthesisof compound 88.

Compound 88: sodium(20S,23S)-28-(2-allyloxy-2-carboxyethyl)-20-((4-((2R,3R)-3-((S)-1-((3S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13-en-16-yl)ethyl)oxiran-2-yl)benzyl)carbamoyl)-23-isopropyl-14,22,25,29,32-pentaoxo-2,5,8,11-tetraoxa-15,21,24,28,33-pentaazapentatriacontane-35-sulfonate

To a solution of compound 62 (50 mg, 43.7 μmol) in DCM (4 mL) was added,dropwise under Ar, the reaction medium containing compound 87 (23.06 mg,43.7 μmol). The reaction medium was stirred at RT for 15 min,concentrated in vacuo and purified by flash chromatography on 15 g ofsilica gel (gradient elution DMC/MeOH/H2O) to give 26 mg of compound 88as a white lacquer (38%).

Example 42: sodium(20S,23S)-28-(2-carboxyethyl)-20-((4-((2R,3R)-3-((S)-1-((3S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13-en-16-yl)ethyl)oxiran-2-yl)benzyl)carbamoyl)-23-isopropyl-14,22,25,29,32-pentaoxo-2,5,8,11-tetraoxa-15,21,24,28,33-pentaazapentatriacontane-35-sulfonate

To a solution of compound 88 (14 mg, 9 μmol) in THF were added dimedone(2.57 mg, 18.0 μmol) and tetrakis(triphenylphosphine)palladium (0) (5.3mg, 4.5 μmol). The reaction medium was stirred for 1 h at RT then wereadded dimedone (2.57 mg, 18.0 μmol) andtetrakis(triphenyl-phosphine)palladium (0) (5.3 mg, 4.5 μmol) andstirring was carried on at RT overnight. At that time, were addeddimedone (2.57 mg, 18.0 μmol) and tetrakis(triphenyl-phosphine)palladium(0) (5.3 mg, 4.5 μmol). The reaction medium was stirred for 4 h at RT,filtered, concentrated in vacuo and purified by flash chromatography on4 g of silica gel (gradient elution DCM/MeOH/H₂O) to give 7 mg ofexample 42 as a white lacquer (51%).

RMN ¹H (500 MHz, δ in ppm, DMSO-d6): 50:50 conformer mixture; 0.78 (d,J=6.8 Hz, 6H); 0.81 (d, J=6.8 Hz, 6H); 0.86 (m, 6H); 1.00 (s, 3H); 1.04(d, J=7.1 Hz, 3H); 1.11 (s, 3H); 1.15 to 1.41 (m, 5H); 1.53 to 1.70 (m,4H); 1.80 (m, 1H); 2.00 (m, 1H); 2.11 to 2.58 (partially masked m, 13H);2.70 (m, 2H); 2.92 to 3.05 (m, 5H); 3.23 (s, 3H); 3.25 to 3.58(partially masked m, 23H); 3.81 (s, 3H); 3.88 (d, J=2.0 Hz, 1H); 4.10 to4.32 (m, 5H); 4.91 (dd, J=3.8 and 9.9 Hz, 1H); 5.10 (m, 1H); 5.80 (d,J=15.2 Hz, 1H); 6.47 (ddd, J=3.8, 11.4 and 15.2 Hz, 1H); 7.06 (d, J=8.6Hz, 1H); 7.18 (dd, J=2.4 and 8.6 Hz, 1H); 7.20 to 7.32 (m, 6H); 7.67 to7.93 (m, 3H); 8.18 to 8.60 (m, 4H). LCMS (E): ES m/z=747.5 [M+2H]²⁺;m/z=1491 [M−H]⁻; m/z=1493 [M+H]⁺; t_(R)=4.74 min.

Synthesis of Example 43: sulfo-Val-GlucoseGln-C52 Benzylic Amine

Compound 89: Sodium(6S,9S)-14-(3-(allyloxy)-3-oxopropyl)-6-((4-((2R,3R)-3-((S)-1-((3S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13-en-16-yl)ethyl)oxiran-2-yl)benzyl)carbamoyl)-9-isopropyl-3,8,11,15,18-pentaoxo-1-((2R,3S,4S,5R,6R)-3,4,5-trihydroxy-6-methoxytetrahydro-2H-pyran-2-yl)-2,7,10,14,19-pentaazahenicosane-21-sulfonate

To a solution of compound 50 (55 mg, 49.9 μmol) in DMF (3 mL) was addeda a solution of compound 87 in DMF (1.66 mL at 30 mM, 49.9 μmol). Thereaction medium was stirred for 18 h at RT, filtered over 0.45 μm,washed twice with DMF (0.8 mL) and purified by reverse phasechromatography on a 5 μm C18 column 30×150 mm (gradient elutionMeCN/0.2N ammonium acetate pH 5.6) to give 19.8 mg of compound 89 as awhite solid (26%).

RMN ¹H (500 MHz, δ in ppm, DMSO-d6): 60:40 conformer mixture; 0.78 (d,J=6.8 Hz, 6H); 0.81 (d, J=6.8 Hz, 6H); 0.81 to 0.87 (m, 6H); 0.99 (s,3H); 1.03 (d, J=7.1 Hz, 3H); 1.12 (s, 3H); 1.30 (m, 1H); 1.52 to 1.62(m, 2H); 1.80 (m, 3H); 1.85 to 2.00 (m, 3H); 2.10 to 2.30 (m, 5H); 2.35to 2.48 (m, 2H); 2.53 (m, 6H); 2.68 (m, 2H); 2.88 to 3.15 (m, 9H); 3.28(m, 3H); 3.38 (m, 3H); 3.40 to 3.58 (m, 5H); 3.80 (s, 3H); 3.87 (d,J=2.3 Hz, 1H); 4.01 (d, J=8.0 Hz, 1H); 4.10 to 4.24 (m, 6H); 4.55 (m,1.2H); 4.57 (0.8H); 4.91 (dd, J=3.8 et 9.9 Hz, 1H); 4.95 (d, J=5.0 Hz,1H); 5.01 (d, J=5.0 Hz, 1H); 5.06 (d, J=5.0 Hz, 1H); 5.10 (m, 1H); 5.18to 5.35 (m, 2H); 5.80 (dd, J=2.0 and 15.2 Hz, 1H); 5.92 (m, 1H); 6.47(ddd, J=3.8, 11.4 and 15.2 Hz, 1H); 7.06 (d, J=8.6 Hz, 1H); 7.18 (dd,J=2.4 and 8.6 Hz, 1H); 7.23 (m, 5H); 7.29 (d, J=2.4 Hz, 1H); 7.69 (t,J=6.8 Hz, 1H); 7.88 (t, J=6.8 Hz, 1H); 7.98 (d, J=9.0 Hz, 0.4H); 8.04(d, J=7.0 Hz, 0.4H); 8.08 (d, J=9.0 Hz, 0.6H); 8.14 (d, J=7.0 Hz, 0.6H);8.32 (t, J=7.0 Hz, 1H); 8.38 (d, J=8.0 Hz, 1H). LCMS (A): ES m/z=746.5[M+2H]²⁺; m/z=1490 [M−H]⁻; m/z=1492 [M+H]⁺; t_(R)=1.4 min.

Example 43: sodium(6S,9S)-14-(2-carboxyethyl)-6-((4-((2R,3R)-3-((S)-1-((3S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13-en-16-yl)ethyl)oxiran-2-yl)benzyl)carbamoyl)-9-isopropyl-3,8,11,15,18-pentaoxo-1-((2R,3S,4S,5R,6R)-3,4,5-trihydroxy-6-methoxytetrahydro-2H-pyran-2-yl)-2,7,10,14,19-pentaazahenicosane-21-sulfonate

To a solution of compound 89 (19 mg, 12.6 μmol) in DMF (1 mL) were addedbarbituric acid (5.94 mg, 37.7 μmol) and a solution of tetrakis(triphenylphosphine)palladium(0) (0.74 mg, 0.63 μmol) in DMF (0.5 mL).The reaction medium was stirred for 3 h at RT then added barbituric acid(5.94 mg, 37.7 μmol) and a solution oftetrakis(triphenylphosphine)palladium(0) (0.74 mg, 0.63 μmol) in DMF(0.5 mL). The reaction medium was stirred at RT overnight, diluted withDMF, filtered over 0.45 μm and purified by two consecutive reverse phasechromatographies on a 5 μm C18 column 30×150 mm (gradient elutionMeCN/0.2N ammonium acetate pH 5.6) to give 4.7 mg of example 43 as awhite solid (25%).

RMN ¹H (500 MHz, δ in ppm, DMSO-d6): 0.80 (m, 6H); 0.85 (m, 6H); 0.99(s, 3H); 1.03 (d, J=7.3 Hz, 3H); 1.11 (s, 3H); 1.33 (m, 1H); 1.58 (m,2H); 1.78 (m, 2H); 1.90 (m, 2H); 2.03 to 2.55 (partially masked m, 11H);2.70 (m, 2H); 2.90 to 3.58 (partially masked m, 19H); 3.18 (s, 3H); 3.81(s, 3H); 3.88 (s, 1H); 4.00 to 4.40 (m, 8H); 4.90 (m, 1H); 4.99 (m, 1H);5.80 (d, J=15.2 Hz, 1H); 6.46 (ddd, J=3.8, 11.4 and 15.2 Hz, 1H); 7.05(d, J=8.6 Hz, 1H); 7.18 (dd, J=2.4 and 8.6 Hz, 1H); 7.22 (m, 4H); 7.29(d, J=2.4 Hz, 1H); 7.31 (m, 1H); 8.30 to 8.70 (m, 5H). LCMS (A): ESm/z=1448 [M−H]⁻; m/z=1450 [M+H]⁺; t_(R)=1.58 min.

For illustrative purposes, two conjugates derived from WO2011/001052,ADC1 and ADC2 depicted below, and one conjugate described inPCT/EP2016/076603, ADC3 depicted below, were also tested.

Physico-Chemical Results

The conjugates of formula (V) were subjected to stability studiesevaluating aggregation (looking at monomeric purity) and loss of DARupon removal of the organic co-solvent used in the formulation and underaccelerated stress conditions (10 day heating at 40° C.).

Stability in the Absence of NMP

ADC stored in 10 mM phosphate 140 mM NaCl pH6.5 5% NMP were filtered ona SEC column to remove the NMP, formulated in 10 mM phosphate 70 mM NaCl5% sucrose pH6.5 and analyzed by HRMS and SEC HPLC to determine the DARand the monomeric purity.

TABLE I 5% NMP no NMP Δ % % % mono- mono- mono- ΔDAR mer protein DAR merDAR mer (%) (%) recovery ADC1 4.1 99 3.25 88.7  −21%  −10% quant. ADC33.5 99.7 3.5 99.5    0% −0.2% n.d. Ex.3   3.4 99.8 3.2 99.7   −6% −0.1%88% Ex.6   4.05 99 3.4 99  −16%    0% 75% Ex.16* 2.5 99.8 2.3 99.8   −8%   0% 73% Ex.19  2.8 97.9 2.4 97.8  −14% −0.1% 72% Ex.23* 2.9 100 2.4100  −17%    0% 76% Ex.26  3.9 100 3.9 100    0%    0% n.d. Ex.29  498.4 3.9 100 −2.5%    0% 76% Ex.32  2.8 95.1 2.9 94.1 0 − 11% 82% *After10 months at 4° C., significant aggregation was observerd for Ex.16 andEx.23 (respectively −22% and −19% in terms of monomeric purity),therefore both batches were purified by gel filtration on Superdex 200pg prior to performing stability studies.

Stability after 10 Days at 40° C. in Phosphate Buffer

ADC in 10 mM phosphate 70 mM NaCl 5% sucrose pH6.5 were heated at 40° C.during 10 days and analyzed by HRMS and SEC HPLC to determine the DARand the monomeric purity.

TABLE 11 After 10 d At t0 at 40° C. Δ % % % mono- mono- mono- ΔDAR merprotein DAR mer DAR mer (%) (%) recovery ADC1 3.25 88.7 3.0 85.7%   −8% −3% 56% ADC3 3.5 99.5 3 85  −14% −15% 46% Ex.3  3.2 99.7 2.9 92   −9% −8% 96% Ex.6  3.4 99 3.3 97   −3%  −2% 91% Ex.16 2.3 99.8 1.8 93.8 −22%  −6% 89% Ex.19 2.4 97.8 1.8 92.9  −25%  −5% quant. Ex.23 2.4 1002.4 91.6    0%  −8% 91% Ex.26 3.9 100 3.6 100   −8%   0% n.d. Ex.29 3.9100 3.9 98.7    0%  −1% 97% Ex.32 2.9 94.1 2.7 89.8   −7%  −5% 95% Ex.413.3 97.9 3.05 75.3 −7.5% −23% quant.

Evaluation of Drug Release after Treatment of the Conjugates of Formula(V) by Cathepsin B

Drug release by Cathepsin B was evaluated in vitro by incubating the ADC(1.5 μg/mL) in the presence of human Cathepsin B (Calbiochem #219362, 40μg/mL) in acetate buffer pH 5 at 37° C. for 24 h and detecting the drugby UPLC-MS-SIR-DAD-ELSD. The slope was determined based on the kineticsover the 1^(st) hour, the half-life on the 24 h kinetics.

TABLE III slope t_(1/2) (h) ADC1 34.3 2 ADC2 14.1 15 Ex.16 12.8 >24Ex.19 22.3 3.5 Ex.23 19.3 6 Ex.32 33.5 2

Cryptophycin conjugates of formula (V) were cleaved by human Cathepsin Bwith a kinetics similar to the one of reference ADCs.

Pharmacological Results

The conjugates of formula (V) were subjected to pharmacological testsfor determining their antitumoral effect.

Evaluation of the Inhibition of Proliferation of the MDA-MB-231 CellLine by the Conjugates of Formula (V)

MDA-MB-231 cells in their exponential growth phase were trypsinized andresuspended in their culture medium (DMEM/F12 Gibco #21331, 10% FCSGibco #10500-056, 2 nM glutamine Gibco #25030). The cell suspension wasseeded in Cytostar 96-well culture plates (GE Healthcare Europe, #RPNQ0163) in the whole culture medium containing serum at a density of5000 cells/well. After incubation for 4 h, successive dilutions of theADC were added to the wells at concentrations decreasing from 10⁻⁷ to10⁻¹² M (in triplicate for each concentration). The cells were culturedat 37° C. in an atmosphere containing 5% CO₂ in the presence of the ADCfor 3 d. On the 4^(th) day, 10 μL of a ¹⁴C-thymidine solution (0.1μCi/well, Perkin Elmer # NEC56825000) were added to each well. Theincorporation of ¹⁴C-thymidine was measured 96 h after the start of theexperiment with a microbeta radioactivity counter (Perkin Elmer). Thedata were expressed in the form of a percentage of survival bydetermining the ratio between the reduced count obtained with the cellstreated with the ADC and the count obtained with the cells of thecontrol wells (treated with the culture medium alone). In certainexperiments, the naked antibody was added to the wells at aconcentration of 1 μM at the start of the experiment and the inhibitionof proliferation was measured as described previously.

TABLE IV IC₅₀ (pM), MDA-MB-231 in the presence of ADC naked Selectivityalone antibody ratio ADC1 42 4571 109 ADC2 46 9377 204 Ex.3 19 5602 295Ex.6 22 7417 337 Ex.16 39 13908 357 Ex.19 29 18243 629 Ex.23 37 9022 243Ex.26 27 33689 1248 Ex.29 67 >100000 >1493 Ex.32 25 7953 318 Ex.35 2511272 451 Ex.41 34 5043 148

Cryptophycin conjugates of formula (V), as well as ADC1 and ADC2, werefound to inhibit the proliferation of MDA-MB-231 cell line with IC₅₀ranging from 19 μM to 67 μM and selectivity ratio ADC alone vs ADC+nakedantibody between 109 and >1493.

Determination of the MTD of the Conjugates of Formula (V) FollowingSingle i.v. Administration in SCID Mice

MTD was determined as the maximal dose that does not induce 15% bodyweight loss during 3 consecutive days for an individual mouse or 20%body weight loss during 1 day or mortality. It was evaluated after asingle intravenous (i.v.) bolus injection in 3 female SCID mice andduring a period of 28 days post-treatment.

TABLE V MTD (mg/kg) ADC1 20 ADC2 30 Ex.3 30 Ex.6 >40 Ex.16 n.d. Ex.19n.d. Ex.23 n.d. Ex.26 30 Ex.29 40 Ex.32 n.d. Ex.35 n.d. Ex.41 30

Tested cryptophycin conjugates of formula (V) displayed MTD in SCID miceranging from 30 mg/kg to ≥40 mg/kg.

Evaluation of the In Vivo Antitumor Activity of Conjugates of Formula(V) Against MDA-MB-231 in SCID Mice Following Single i.v. Administration

In vivo antitumor activity was evaluated at 3 dose-levels againstmeasurable breast MDA-MB-231 xenografts implanted s.c. in female SCIDmice. Control groups were left untreated. Conjugates were administeredby a single i.v. bolus injection, the day of the treatment was indicatedon each graph by an arrow (▾).

For the evaluation of antitumor activity of conjugates, animals wereweighed twice weekly and tumors were measured twice weekly by caliper.Animal body weights included the tumor weights. Tumor volume werecalculated using the formula mass (mm³)=[length (mm)×width (mm)²]/2. Theprimary efficacy end points were ΔT/ΔC, percent median regression,partial and complete regressions (PR and CR). Changes in tumor volumefor each treated (T) and control (C) were calculated for each tumor bysubtracting the tumor volume on the day of first treatment (staging day)from the tumor volume on the specified observation day. The median ΔTwas calculated for the treated group and the median ΔC was calculatedfor the control group. Then the ratio ΔT/ΔC was calculated and expressedas a percentage: ΔT/ΔC=(delta T/delta C)×100.

The percentage of tumor regression was defined as the % of tumor volumedecrease in the treated group at a specified observation day (t)compared to its volume on the first day of first treatment (t0). At aspecific time point and for each animal, % regression was calculated.The median % regression was then calculated for the group. % regression(at t)=((Volume_(t0)−Volume_(t))/Volume_(t0))×100. Regressions weredefined as partial (PR) if the tumor volume decreased to 50% of thetumor volume at the start of treatment and complete (CR) when tumorvolume cannot be measured (0 mm³). Tumor free survivor (TFS) was definedas the animals with undetectable tumors at the end of the study (>100days post last treatment).

Evaluation of the In Vivo Antitumor Activity of Ex. 6 Against MDA-MB-231in SCID Mice Following Single i.v. Administration

TABLE VI Dose Median ΔT/ΔC Median % of Regressions (mg/kg) in % (D36)regression PR CR TFS Control — — — — — — ADC2 2.5 8 — 1 1 1 Ex.6 5 <0100% 5 4 — 2.5 <0  4% 2 1 — 1.25 33 — — — —

Evaluation of the In Vivo Antitumor Activity of Ex. 16, Ex. 19, Ex. 23and Ex. 32 Against MDA-MB-231 in SCID Mice Following Single i.v.Administration at 2.5 mg/kg

TABLE VII Dose Median ΔT/ΔC Median % of Regressions (mg/kg) in % (D59)regression PR CR TFS Control — — — — — — ADC2 2.5 <0  14% — — — Ex.162.5 <0  92% 6 3 — Ex.19 2.5 <0 100% 6 5 2 Ex.23 2.5 <0 100% 6 6 1 Ex.322.5 <0 100% 6 6 2

Evaluation of the In Vivo Antitumor Activity of Ex. 16, Ex. 19, Ex. 23and Ex. 32 Against MDA-MB-231 in SCID Mice Following Single i.v.Administration at 1.25 mg/kg

TABLE VIII Dose Median ΔT/ΔC Median % of Regressions (mg/kg) in % (D50)regression PR CR TFS Control — — — — — — ADC2 1.25 14 — — — — Ex.16 1.257 — — — — Ex.19 1.25 1 — 1 1 — Ex.23 1.25 0 — 1 — — Ex.32 1.25 9 — 2 1 —

Evaluation of the In Vivo Antitumor Activity of Ex. 26 AgainstMDA-MB-231 in SCID Mice Following Single i.v. Administration

TABLE IX Dose Median ΔT/ΔC Median % of Regressions (mg/kg) in % (D36)regression PR CR TFS Control — — — — — — ADC2 2.5 8 — 1 — 1 Ex.26 5 <0100% 6 6 2 2.5 <0 100% 6 4 1 1.25 10 — 1 — —

Evaluation of the In Vivo Antitumor Activity of Ex. 29 AgainstMDA-MB-231 in SCID Mice Following Single i.v. Administration

TABLE X Dose Median ΔT/ΔC Median % of Regressions (mg/kg) in % (D27)regression PR CR TFS Control — — — — — — Ex.29 4 <0 100% 6 6 6 2 <0 100%6 6 4 1 0 — — — —

Evaluation of the In Vivo Antitumor Activity of Ex. 35 AgainstMDA-MB-231 in SCID Mice Following Single i.v. Administration

TABLE XI Dose Median ΔT/ΔC Median % of Regressions (mg/kg) in % (D27)regression PR CR TFS Control — — — — — — Ex.35 4 <0 100% 6 6 4 2 <0 100%6 4 1 1 3 — — — —

Evaluation of the In Vivo Antitumor Activity of Ex. 41 AgainstMDA-MB-231 in SCID Mice Following Single i.v. Administration

TABLE XII Dose Median ΔT/ΔC Median % of Regressions (mg/kg) in % (D49)regression PR CR TFS Control — — — — — — Ex.26 4 <0 100% 6 6 6 2 <0 100%6 6 4 1 <0  51% 3 2 1 Ex.41 4 <0 100% 6 4 2 2 <0  25% 2 — — (D46) 1 20 —— — —

All tested cryptophycin ADC of the invention, as well as ADC2, displayedantitumor activity at doses ranging from 1 mg/kg to 5 mg/kg.

It is therefore apparent that the compounds (V) of the invention have ananticancer activity.

Accordingly, in another of its aspects, the invention also relates tothe use of cryptophycin conjugates of formula (V) as anticancer agents.

Accordingly, in another of its aspects, the invention also relates tothe use of cryptophycin conjugates of formula (V) for the preparation ofa medicament for treating cancer, for instance breast cancer.

The present invention, according to another of its aspects, alsoprovides conjugates of formula (V) according to the present inventionfor use in the treatment of cancer.

The present invention, according to another of its aspects, alsoprovides medicaments which comprise at least one conjugate of formula(V).

These medicaments are employed therapeutically, especially in thetreatment of cancer, for instance breast cancer.

According to another of its aspects, the present invention relates topharmaceutical compositions comprising as active principle a conjugateof formula (V) according to the invention. These pharmaceuticalcompositions comprise an effective dose of at least one conjugate offormula (V) according to the invention and also at least onepharmaceutically acceptable excipient.

Thus, according to another aspect, the present invention relates to apharmaceutical composition comprising at least one conjugate of formula(V) according to the present invention, and also at least onepharmaceutically acceptable excipient.

The said excipients are selected, in accordance with the pharmaceuticalform and method of administration desired, from the customaryexcipients, which are known to a person skilled in the art.

The present invention, according to another of its aspects, alsoprovides a method of treating the pathologies indicated above such ascancer, for instance breast cancer, which comprises administering to apatient an effective dose of a conjugate of formula (V) according to theinvention.

1. A conjugate of formula (V):

wherein: R₁ represents a (C₁-C₆)alkyl group; R₂ and R₃ represent,independently of each other, a hydrogen atom or a (C₁-C₆)alkyl group; oralternatively R₂ and R₃ form, together with the carbon atom to whichthey are attached, a (C₃-C₆)cycloalkyl or a (C₃-C₆)heterocycloalkylgroup; R₄ and R₅ represent, independently of each other, a hydrogenatom, a (C₁-C₆)alkyl group, a (C₁-C₆)alkyl-NH(R₁₂) group, a(C₁-C₆)alkyl-OH group, a (C₁-C₆)alkyl-SH group, or a (C₁-C₆)alkyl-CO₂Hgroup; or alternatively R₄ and R₅ form together with the carbon atom towhich they are attached a (C₃-C₆)cycloalkyl or a (C₃-C₆)heterocycloalkylgroup; X represents O or N(R₆); R₆ represents a hydrogen atom or a(C₁-C₆)alkyl group; R₇ and R₈ represent, independently of each other, ahydrogen atom, a (C₁-C₆)alkyl group, a (C₁-C₆)alkyl-CO₂H group or a(C₁-C₆)alkyl-N(C₁-C₆)alkyl₂ group; or alternatively R₇ and R₈ formtogether with the carbon atom to which they are attached a(C₃-C₆)cycloalkyl group or a (C₃-C₆)heterocycloalkyl group; R₉represents at least one substituent of the phenyl nucleus chosen, eachindependently of each other, from a hydrogen atom, —OH, (C₁-C₄)alkoxy, ahalogen atom, —NH₂, —NH(C₁-C₆)alkyl or —N(C₁-C₆)alkyl₂ or—NH(C₁-C₆)cycloalkyl or (C₃-C₆)heterocycloalkyl group; R₁₀ represents atleast one substituent of the phenyl nucleus chosen from a hydrogen atomand a (C₁-C₄)alkyl group; Y represents NR₁₁—(C₁-C₆)alkyl-,—O—(C₁-C₆)alkyl-, or —S—(C₁-C₆)alkyl-; R₁₁ and R₁₂ represent,independently of each other, a hydrogen atom or (C₁-C₆)alkyl; L isdefined as in formula (I):RCG1-L-P  (I) wherein RCG1 is selected from a R_(a)Z_(a)—C(═O) reactivegroup wherein Z_(a) represents a single bond, —O— or —NH—, and R_(a)represents a hydrogen atom, a (C₁-C₆)alkyl group, a (C₃-C₇)cycloalkylgroup, an alkenyl group, an aryl group, a heteroaryl group or a(C₃-C₇)heterocycloalkyl group, the aryl group, the heteroaryl groupand/or the (C₃-C₇)heterocycloalkyl group being optionally substituted by1 to 5 groups selected from a halogen atom, an alkyl group, an alkoxygroup, a hydroxyl group, an oxo group, a nitro group and a cyano group;or wherein RCG1 is selected from a maleimido

group, a haloacetamido,

Cl—; N₃—, HO—, HS—,

H₂N—, HC≡C— group or an activated C≡C group, an O-alkyl hydroxylamine,or a Pictet-Spengler reaction substrate; and wherein R₂₁ represents ahydrogen atom or a (C₁-C₆)alkyl group; P represents a hydrogen atom, —OHor an activated O; L represents a linker of formula (II):

wherein: L1 is of formula (III):

wherein: when P represents a hydrogen atom, then x=0 or 1 and y=1 andz=0; when P represents —OH, then x=y=z=0; when P represents an activatedO, then x=1 and y=z=0, or x=y=z=1; J₁, J₂, J₃ and J₄ are chosen,independently of each other, from CA₁ and N; ALK represents a(C₁-C₁₂)alkylene group; A₁, A₂, A₃, A₄, A₅, and A₆ represent,independently of each other, a hydrogen atom or a (C₁-C₆)alkyl group;(AA)w represents a sequence of w substituted amino acids (AA_(s)) ornon-substituted amino acids (AA_(ns)) connected together via peptidebonds; w represents an integer ranging from 2 to 12; wherein: (AA)wcontains at least one substituted amino acid (AA_(s)), and L2 representsa single bond, a (C₁-C₆)alkyl group, a (C₁-C₆)alkyl-(OCH₂CH₂)_(i) group,a (C₁-C₆)alkyl-(OCH₂CH₂)_(i)—O(C₁-C₆)alkyl group, a(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, a CH(SO₃H)—(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-CH(SO₃H) group, a (C₁-C₆)alkyl-cyclohexyl group, aC(═O)—(C₁-C₆)alkyl group, a C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—O(C₁-C₆)alkyl group, aC(═O)—(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, a C(═O)—CH(SO₃H)—(C₁-C₆)alkylgroup, a C(═O)—(C₁-C₆)alkyl-CH(SO₃H) group, aC(═O)—(C₁-C₆)alkyl-cyclohexyl group, a NA₈-(C₁-C₆)alkyl group, aNA₈-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aNA₈-(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—O(C₁-C₆)alkyl group, aNA₈-(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, a NA₈-(C₁-C₆)alkyl-CH(SO₃H) group,a C(═O)—NA₈-(C₁-C₆)alkyl group, a C(═O)—NA₈-(C₁-C₆)alkyl-(OCH₂CH₂)_(i)group, a C(═O)—NA-(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—O(C₁-C₆)alkyl group, aC(═O)—NA₈-(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-CH(SO₃H) group, a NA₇-(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-NA₇ group, a NA₇-(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, aNA₇-aryl group, a NA₇-heteroaryl group, a(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alkyl group, aC(═O)—NA₇-(C₁-C₆)alkyl group, a C(═O)—(C₁-C₆)alkyl-NA₇ group, aC(═O)—NA₇-(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, a C(═O)—NA₇-aryl group, aC(═O)—NA₇-heteroaryl group, a C(═O)—(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkylgroup, a C(═O)—(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, aC(═O)—(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, aC(═O)—(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alkyl group, aNA₈-(C₁-C₆)alkyl-NA₇ group, a NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkylgroup, a NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aNA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, aNA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aNA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, aNA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aNA₈-(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇ group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—NA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group or aC(═O)—NA₈-(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alkyl group; A₇represents a straight or branched, saturated or unsaturated, optionallysubstituted C₁-C₁₆₀ hydrocarbon chain wherein optionally at least onemethylene unit is independently replaced by —NHC(═O)—, —N(alkyl)C(═O)—,—C(═O)NH—, —C(═O)N(alkyl)-, —OC(═O)—, —C(═O)O—, —OC(═O)O—, —CH(OH)—,—CH(SO₃H)—, —O—, —C(═O)—, —S(═O)—, —S(═O)₂—, —NHS(═O)₂—,—N(alkyl)S(═O)₂—, —S(═O)₂NH—, —S(═O)₂N(alkyl)-, —P(═O)(OH)—,—P(═O)(OH)O—, —O—P(═O)(OH)—, —O—P(═O)(OH)—O— or a heterocycloalkyl groupoptionally substituted with at least one substituent, identical ordifferent, chosen from —OH, —Oalkyl, -alkyl, a halogen atom, —NH₂,—NHalkyl, and —N(alkyl)₂, wherein —CH(SO₃H)— is optionally the alkalimetal salt; A₈ represents a hydrogen atom or a (C₁-C₆)alkyl group; and irepresents an integer ranging from 1 to 50, wherein said substitutedamino acid (AA_(s)) is of formula (VI):

wherein: T represents a saturated or unsaturated, linear or branched,(C₁-C₈) trivalent alkyl group; U group represents a single bond,—NHC(═O)—, —N(alkyl)C(═O)—, —C(═O)NH—, —C(═O)N(alkyl)-, —NHC(═O)NH—,—NHC(═NH)NH—, —OC(═O)—, —C(═O)O—, —OC(═O)O—, —S—, —Se—, —O—, —NH—,—N(alkyl)-, —C(═O)—, —OP(═O)—, —S(═O)—, —S(═O)₂—, —NHS(═O)₂—,—N(alkyl)S(═O)₂—, —S(═O)₂NH—, —S(═O)₂N(alkyl)-, —P(═O)(OH)—,—P(═O)(OH)O—, —O—P(═O)(OH)—, or —O—P(═O)(OH)—O; A₉ represents a straightor branched, saturated or unsaturated, optionally substituted C₁-C₁₆₀hydrocarbon chain wherein optionally at least one methylene unit isindependently replaced by —NH—C(═O)—, —N(alkyl)C(═O)—, —C(═O)NH—,—C(═O)N(alkyl)-, —NHC(═O)NH—, —NHC(═NH)NH—, —OC(═O)—, —C(═O)O—,—OC(═O)O—, —CH(OH)—, —CH(SO₃H)—, —CH(Oalkyl)-, —CHF—, —CF₂—, —S—, —Se—,—O—, —NH—, —N(alkyl)-, —N⁺H(alkyl)-, —N(alkyl)₂-, —C(═O)—, —OP(═O)—,—S(═O)—, —S(═O)₂—, —NHS(═O)₂—, —N(alkyl)S(═O)₂—, —S(═O)₂NH—,—S(═O)₂N(alkyl)-, —P(═O)(OH)—, —P(═O)(OH)O—, —O—P(═O)(OH)—,—O—P(═O)(OH)—O— or a heterocycloalkyl group optionally substituted withat least one substituent, identical or different, chosen from —OH,—Oalkyl, -alkyl, a halogen atom, —NH₂, —NHalkyl, and —N(alkyl)₂; andwherein —CH(SO₃H)— is optionally the alkali metal salt and G representsthe product of reaction between RCG1 and RCG2, wherein RCG2 is anorthogonal reactive chemical group present on a polypeptide such as theantibody (Ab); and Ab represents an antibody.
 2. The conjugate offormula (V) according to claim 1, having the following structure:

wherein: R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁, R₁₂, X, Y, L, G,RCG1, RCG2 and Ab are as in formula (V) according to claim
 1. 3. Theconjugate of formula (V) according to claim 1, wherein RCG2 is selectedfrom the ε-amino groups of lysines borne by the side chains of thelysine residues that are present at the surface of an antibody, one theα-amino groups of N-terminal amino acids of antibody heavy and lightchains, the saccharide groups of the hinge region, the thiols ofcysteines generated by reducing intra-chain disulfide bonds or thethiols of engineered cysteines, the amide groups borne by the sidechains of some glutamine residues that are present at the surface of anantibody, and aldehyde groups introduced using formylglycine generatingenzyme.
 4. The conjugate of formula (V) according to claim 1, wherein:when RCG1 represents a N-hydroxysuccinimidyl ester, RCG2 represents aNH₂ group, or when RCG1 represents a maleimido function, a haloacetamidofunction, a chlorine atom or an activated disulfide, RCG2 represents a—SH group, or when RCG1 represents a N₃ group, RCG2 represents a C≡CHgroup or an activated C≡C group such as a cyclooctyne moiety, or whenRCG1 represents a OH or NH₂ group, RCG2 represents a carboxylic acid oramide function, or when RCG1 represents a SH group, RCG2 represents amaleimido function, a haloacetamido function or an activated disulfidefunction, or when RCG1 represents a C≡CH group or an activated C≡Cgroup, RCG2 represents a N₃ group, or when RCG1 represents a O-alkylhydroxylamine function or a Pictet-Spengler reaction substrate, RCG2represents an aldehyde or ketone function.
 5. The conjugate of formula(V) according to claim 1, wherein G is selected from the groupconsisting of:


6. The conjugate of formula (V) according to claim 1, wherein Trepresents

U represents —NH—C(═O)—, or —C(═O)NH—; A₉ represents—[(CH₂)₂—O]_(b)—CH₃; and wherein b represents an integer ranging from 1to
 50. 7. The conjugate of formula (V) according to claim 1, wherein thesequence (AA_(s))w containing at least one substituted amino acid(AA_(s)), is selected from the list:


8. The conjugate of formula (V) according to claim 1, wherein (AA)wcontains at least one substituted amino acid (AA_(s)) and L2 representsa (C₁-C₆)alkyl group, a —C(═O)—(C₁-C₆)alkyl group, or a(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group; wherein A₇ is a—C(═O)—(CH₂)₂—C(═O)—NH—(CH₂)₂—SO₃H group; and wherein —CH(SO₃H)— isoptionally the alkali metal salt.
 9. The conjugate of formula (V)according to claim 1, wherein A₇ represents a —C(═O)—[(CH₂)₂—O]₄—CH₃group, a —C(═O)—[(CH₂)₂—O]₇—CH₃ group, a —C(═O)—[(CH₂)₂—O]₂₄—CH₃ group;a —C(═O)—(CH₂)₂—C(═O)—NH—(CH₂)₂—SO₃H group, a—C(═O)—(CH₂)₂—C(═O)—NH—[(CH₂)₂—O-]₄-(CH₂)₂—C(═O)—NH—(CH₂)₂—SO₃H group;and wherein —CH(SO₃H)— is optionally the alkali metal salt.
 10. Theconjugate of formula (V) according to claim 1, wherein (AA)w contains atleast one substituted amino acid (AA_(s)) optionally a w non-substitutedamino acid (AA_(ns)) and L2 represents: a NA₇-(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-NA₇ group, a NA₇-(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, aNA₇-aryl group, a NA₇-heteroaryl group, a(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, a(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, a(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alkyl group, aC(═O)—NA₇-(C₁-C₆)alkyl group, a C(═O)—(C₁-C₆)alkyl-NA₇ group, aC(═O)—NA₇-(CH₂CH₂O)_(i)(C₁-C₆)alkyl group, a C(═O)—NA₇-aryl group, aC(═O)—NA₇-heteroaryl group, a C(═O)—(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkylgroup, a C(═O)—(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, aC(═O)—(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, aC(═O)—(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alkyl group, aNA₈-(C₁-C₆)alkyl-NA₇ group, a NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkylgroup, a NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aNA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, aNA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aNA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, aNA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aNA₈-(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alky group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇ group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇C(═O)—(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—NA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-C(═O)NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl group, aC(═O)—NA₈-(C₁-C₆)alkyl-NA₇-(C₁-C₆)alkyl-(OCH₂CH₂)_(i) group or aC(═O)—NA₈-(C₁-C₆)alkyl-(OCH₂CH₂)_(i)—NA₇-(C₁-C₆)alkyl group.
 11. Theconjugate of formula (V) according to claim 1, wherein (AA)w contains atleast one substituted amino acid (AA_(s)) optionally a w non-substitutedamino acid (AA_(ns)) and L2 represents:


12. The conjugate of formula (V) according to claim 1, wherein (AA)wcontains at least one substituted amino acid (AA_(s)) optionally a wnon-substituted amino acid (AA_(ns)) and L2 comprises an A₇ representinga C(═O)—[(CH₂)₂—O]₄—CH₃ group, a C(═O)—[(CH₂)₂—O]₇—CH₃ group, aC(═O)—[(CH₂)₂—O]₂₄—CH₃ group, a C(═O)—(CH₂)₂—C(═O)—NH—(CH₂)₂—SO₃H group,a C(═O)—(CH₂)₂—C(═O)—NH—[(CH₂)₂—O]₄—(CH₂)₂—C(═O)—NH—(CH₂)₂—SO₃H group;and wherein —CH(SO₃H)— is optionally the alkali metal salt.
 13. Theconjugate of formula (V) according to claim 1, wherein L is selectedfrom the following list:


14. The conjugate of formula (V) according to claim 1, wherein: U grouprepresents a single bond, —NHC(═O)—, —N(alkyl)C(═O)—, —C(═O)NH—,—C(═O)N(alkyl)-, —NH—, —N(alkyl)-, —C(═O)—, A₉ represents a straight orbranched, saturated or unsaturated, optionally substituted C₁-C₁₆₀hydrocarbon chain wherein optionally at least one methylene unit isindependently replaced by —NH—C(═O)—, —N(alkyl)C(═O)—, —C(═O)NH—,—C(═O)N(alkyl)-, —NH—, —N(alkyl)-, —C(═O)—, —CH(OH)—, —CH(SO₃H)—, —O—,or a heterocycloalkyl group optionally substituted with at least onesubstituent, identical or different, chosen from —OH, —Oalkyl, -alkyl,and a halogen atom; and wherein —CH(SO₃H)— is optionally the alkalimetal salt.
 15. The conjugate of formula (V) according to claim 1, whichis selected from the following list:

wherein Ab represents an antibody.
 16. The conjugate of formula (V)according to claim 1, wherein Y is positioned in an ortho (o), meta (m)or para (p) position of the phenyl nucleus and represents a—NR₁₁—(CH₂)_(n)— group in which n represents an integer ranging from 1to
 6. 17. A process for preparing a conjugate of formula (V) as definedin claim 1 comprising the steps of: (i) placing in contact and leavingto react: an optionally buffered aqueous solution of an antibody,optionally modified by means of a modifying agent, and a solution of acryptophycin payload of formula (IV):

wherein: R₁ represents a (C₁-C₆)alkyl group; R₂ and R₃ represent,independently of each other, a hydrogen atom or a (C₁-C₆)alkyl group; oralternatively R₂ and R₃ form, together with the carbon atom to whichthey are attached, a (C₃-C₆)cycloalkyl or a (C₃-C₆)heterocycloalkylgroup; R₄ and R₅ represent, independently of each other, a hydrogenatom, a (C₁-C₆)alkyl group, a (C₁-C₆)alkyl-NH(R₁₂) group, a(C₁-C₆)alkyl-OH group, a (C₁-C₆)alkyl-SH group, or a (C₁-C₆)alkyl-CO₂Hgroup; or alternatively R₄ and R₅ form together with the carbon atom towhich they are attached a (C₃-C₆)cycloalkyl or a (C₃-C₆)heterocycloalkylgroup; X represents O or N(R₆); R₆ represents a hydrogen atom or a(C₁-C₆)alkyl group; R₇ and R₈ represent, independently of each other, ahydrogen atom, a (C₁-C₆)alkyl group, a (C₁-C₆)alkyl-CO₂H group or a(C₁-C₆)alkyl-N(C₁-C₆)alkyl₂ group; or alternatively R₇ and R₈ formtogether with the carbon atom to which they are attached a(C₃-C₆)cycloalkyl group or a (C₃-C₆)heterocycloalkyl group; R₉represents at least one substituent of the phenyl nucleus chosen, eachindependently of each other, a hydrogen atom, —OH, (C₁-C₄)alkoxy, ahalogen atom, —NH₂, —NH(C₁-C₆)alkyl or —N(C₁-C₆)alkyl₂ or—NH(C₁-C₆)cycloalkyl or (C₃-C₆)heterocycloalkyl group; R₁₀ represents atleast one substituent of the phenyl nucleus chosen from a hydrogen atomand a (C₁-C₄)alkyl group; Y represents NR₁₁—(C₁-C₆)alkyl-,—O—(C₁-C₆)alkyl-, or —S—(C₁-C₆)alkyl-; R₁₁ and R₁₂ represent,independently of each other, a hydrogen atom or (C₁-C₆)alkyl; L isdefined as in formula (I) according to claim 1 and represents a linkerof formula (II) as defined in claim 1; and RCG1 represents a reactivechemical group present at the end of the linker L, wherein: the chemicalgroup RCG1 of the cryptophycin payload of formula (IV) is reactivetowards the chemical group RCG2 present on the polypeptide so as toattach the cryptophycin payload of formula (IV) to the antibody byformation of a covalent bond; and (ii) optionally separating theconjugate formed in step (i) from the cryptophycin payload of formula(IV) and/or from the unreacted antibody and/or from any aggregates thatmay have formed.
 18. The process for preparing the conjugate of formula(V) according to claim 17, wherein Y is positioned in an ortho (o), meta(m) or para (p) position of the phenyl nucleus and represents a—NR₁₁—(CH₂)_(n)— group in which n represents an integer ranging from 1to
 6. 19. A pharmaceutical composition comprising a conjugate of formula(V) according to claim 1 and a pharmaceutically acceptable excipient.20. The pharmaceutical composition of claim 19, wherein: U grouprepresents a single bond, —NHC(═O)—, —N(alkyl)C(═O)—, —C(═O)NH—,—C(═O)N(alkyl)-, —NH—, —N(alkyl)-, —C(═O)—, A₉ represents a straight orbranched, saturated or unsaturated, optionally substituted C₁-C₁₆₀hydrocarbon chain wherein optionally at least one methylene unit isindependently replaced by —NH—C(═O)—, —N(alkyl)C(═O)—, —C(═O)NH—,—C(═O)N(alkyl)-, —NH—, —N(alkyl)-, —C(═O)—, —CH(OH)—, —CH(SO₃H)—, —O—,or a heterocycloalkyl group optionally substituted with at least onesubstituent, identical or different, chosen from —OH, —Oalkyl, -alkyl,and a halogen atom; and wherein —CH(SO₃H)— is optionally the alkalimetal salt.
 21. A method of treating cancer in a patient in needthereof, comprising administering a therapeutically effective amount ofa conjugate of formula (V) according to claim
 1. 22. The method oftreating cancer of claim 21, wherein: U group represents a single bond,—NHC(═O)—, —N(alkyl)C(═O)—, —C(═O)NH—, —C(═O)N(alkyl)-, —NH—,—N(alkyl)-, —C(═O)—, A₉ represents a straight or branched, saturated orunsaturated, optionally substituted C₁-C₁₆₀ hydrocarbon chain whereinoptionally at least one methylene unit is independently replaced by—NH—C(═O)—, —N(alkyl)C(═O)—, —C(═O)NH—, —C(═O)N(alkyl)-, —NH—,—N(alkyl)-, —C(═O)—, —CH(OH)—, —CH(SO₃H)—, —O—, or a heterocycloalkylgroup optionally substituted with at least one substituent, identical ordifferent, chosen from —OH, —Oalkyl, -alkyl, and a halogen atom; andwherein —CH(SO₃H)— is optionally the alkali metal salt.